Gene therapy for treating mucopolysaccharidosis type ii

ABSTRACT

A suspension useful for AAV9-mediated intrathecal and/or systemic delivery of an expression cassette containing a hIDS gene is provided herein. Also provided are methods and kits containing these vectors and compositions useful for treating Hunter syndrome and the symptoms associated with Hunter syndrome.

STATEMENT OF FEDERALLY FUNDED RESEARCH

This invention was made with government support under grant numbersR01DK54481, P40OD010939, and P30ES013508 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

1. INTRODUCTION

The invention relates to a gene therapy approach for treatingMucopolysaccharidosis Type II (MPS II), also known as Hunter Syndrome.

2. BACKGROUND OF THE INVENTION

MPS II, also known as Hunter syndrome, is a rare X-linked recessivegenetic disease affecting 1 in 100,000 to 1 in 170,000 individuals,primarily males. This progressive and devastating disease is caused bymutations in the IDS gene, leading to deficiency of the lysosomalenzyme, iduronate-2-sulfatase—an enzyme required for the lysosomalcatabolism of heparan sulfate and dermatan sulfate. These ubiquitouspolysaccharides, called GAGs (glycosaminoglycans), accumulate in tissuesand organs of MPS II patients resulting in characteristic storagelesions and diverse disease sequelae. Morbidity and mortality are highin this patient population—in patients with the severe phenotype(characterized by neurocognitive deterioration) death has been reportedto occur at a mean age of 11.7 years; in patients with mild orattenuated phenotype, death has been reported at 21.7 years.

Patients with MPS II appear normal at birth, but signs and symptoms ofdisease typically present between the ages of 18 months and 4 years inthe severe form, and between 4 and 8 years in the attenuated form. Signsand symptoms common to all affected patients include short stature,coarse facial features, macrocephaly, macroglossia, hearing loss,hepato- and splenomegaly, dystosis multiplex, joint contracture, spinalstenosis and carpal tunnel syndrome. Frequent upper respiratory and earinfections occur in most patients and progressive airway obstruction iscommonly found leading to sleep apnea and often death. Cardiac diseaseis a major cause of death in this population and is characterized byvalvular dysfunction leading to right and left ventricular hypertrophyand heart failure. Death is generally attributed to obstructive airwaydisease or cardiac failure.

In severe forms of the disease, early developmental milestones may bemet, but developmental delay is readily apparent by 18-24 months. Somepatients fail hearing screening tests in the first year and othermilestones are delayed, including the ability to sit unsupported,ability to walk, and speech. Developmental progression begins to plateauaround 6.5 years. While half the children with MPS II become toilettrained, most children, if not all, will lose this ability as thedisease progresses.

Patients with significant neurologic involvement exhibit severebehavioral disturbances, including hyperactivity, obstinacy, andaggression beginning in the second year of life and continuing to age8-9, when neurodegeneration attenuates this behavior.

Seizures are reported in over half of severely affected patients whoreach the age of 10, and by the time of death most patients with CNSinvolvement are severely mentally handicapped and require constant care.Although patients with attenuated disease exhibit normal intellectualfunctioning, MRI imaging reveals gross brain abnormalities in allpatients with MPS II including white matter lesions, enlarged ventriclesand brain atrophy.

Enzyme Replacement Therapy (“ERT”) with recombinant idursulfase(Elaprase®, Shire Human Genetic Therapies) is the only approvedtreatment for Hunter syndrome and is administered as a weekly infusion.However, ERT as currently administered does not cross the blood brainbarrier (“BBB”) and is therefore unable to address the unmet need inpatients with severe disease—i.e., MPS II with CNS/neurocognitive andbehavioral involvement. Current efforts to address this issue are aimedat modifying the enzyme to enable it to cross the BBB.

3. SUMMARY OF THE INVENTION

The use of a replication deficient adeno-associated virus (“AAV”) todeliver a human iduronate-2-sulfatase (“hIDS”) gene to the CNS ofpatients (human subjects) diagnosed with MPS II, also known as Hunterssyndrome, is provided herein. The recombinant AAV (“rAAV”) vector usedfor delivering the hIDS gene (“rAAV.hIDS”) should have a tropism for theCNS (e.g., an rAAV bearing an AAV9 capsid), and the hIDS transgeneshould be controlled by specific expression control elements, e.g., ahybrid of cytomegalovirus (CMV) enhancer and the chicken beta actinpromoter (CB7). Pharmaceutical compositions suitable forintrathecal/intracisternal administration comprise a suspension ofrAAV.hIDS vectors in a formulation buffer comprising a physiologicallycompatible aqueous buffer, a surfactant and optional excipients. TherAAV suspension is further characterized in that:

(i) the rAAV Genome Copy (GC) titer is at least 1.0×10¹³ GC/ml (+/−20%);

(ii) the rAAV Empty/Full particle ratio is between 0.01 and 0.05(95%-99% free of empty capsids) as determined by SDS-PAGE analysis (seeExample 5D), or in other embodiments at least about 50%, at least about80%, at least about 85%, or at least about 90%, free of empty capsids;and/or (iii) a dose of at least about 2.5×10¹⁰ GC/g brain mass to about3.6×10¹¹ GC/g brain mass of the rAAV suspension has potency.

Also provided herein is a pharmaceutical composition as provided hereinwhich administrable to a human subject in need thereof by intrathecalinjection. In certain embodiments, use of a pharmaceutical compositioncontaining the rAAV.hIDS described herein are used in preparing amedicament administrable to a human subject in need thereof byintrathecal injection. The human subject (patient) may have beenpreviously diagnosed with mucopolysaccharidosis II (MPS II) or severeHunter syndrome.

Potency can be measured by in vitro cell culture assays, e.g., the invitro potency assay described in Example 5G herein, in which HEK293 orHuh7 cells are transduced with a known multiplicity of rAAV GCs per celland the supernatant is assayed for IDS activity 72 hourspost-transduction using the 4MU-iduronide enzymatic assay.

Such rAAV.hIDS vector preparations can be administered to pediatric oradult human subjects by intrathecal/intracisternal injection to achievetherapeutic levels of hIDS expression in the CNS. Patients who arecandidates for treatment are pediatric and adult patients with severe orattenuated MPS II disease. Severe disease is defined as early-stageneurocognitive deficit with a developmental quotient (DQ) (BSID-III)that is at least 1 standard deviation below the mean or documentedhistorical evidence of a decline of greater than 1 standard deviation onsequential testing.

Therapeutically effective intrathecal/intracisternal doses of therAAV.hIDS for patients with Hunter syndrome range from 1.4×10¹³ to7.0×10¹³ GC (flat doses)—the equivalent of 10¹⁰ to 5×10¹⁰ GC/g brainmass of the patient, or 3.8×10¹² to 7.0×10¹³ GC (flat doses)—theequivalent of 10¹⁰ to 5×10¹⁰ GC/g brain mass of the patient.Alternatively, the following therapeutically effective flat doses can beadministered to patients of the indicated age group:

Newborns: about 3.8×10¹² to about 1.9×10¹⁴ GC;

3-9 months: about 6×10¹² to about 3×10¹⁴ GC;

9-36 months: about 10¹³ to about 5×10¹⁴ GC;

3-12 years: about 1.2×10¹³ to about 6×10¹⁴ GC;

12+ years: about 1.4×10¹³ to about 7.0×10¹⁴ GC;

18+ years (adult): about 1.4×10¹³ to about 7.0×10¹⁴ GC.

In some embodiments, the dose administered to a 12+ year old MPS IIpatient (including 18+ year old) is 1.4×10¹³ genome copies (GC)(1.1×10¹⁰ GC/g brain mass). In some embodiments, the dose administeredto a 12+ year old MPS II patient (including 18+ year old) is 7×10¹³ GC(5.6×10¹⁰ GC/g brain mass). In still a further embodiment, the doseadministered to an MPS II patient is at least about 4×10⁸ GC/g brainmass to about 4×10¹¹ GC/g brain mass. In certain embodiments, the doseadministered to MPS II newborns ranges from about 1.4×10¹¹ to about1.4×10¹⁴ GC; the dose administered to infants 3-9 months ranges fromabout 2.4×10¹¹ to about 2.4×10¹⁴ GC; the dose administered to MPS IIchildren 9-36 months ranges: about 4×10¹¹ to about 4×10¹⁴ GC; the doseadministered to MPS II children 3-12 years: ranges from about 4.8×10¹¹to about 4.8×10¹⁴ GC; the dose administered to children and adults 12+years ranges from about 5.6×10¹¹ to about 5.6×10¹⁴ GC.

The goal of the treatment is to functionally replace the patient'sdefective iduronate-2-sulfatase via rAAV-based CNS-directed gene therapyas a viable approach to treat disease. Efficacy of the therapy can bemeasured by assessing (a) the prevention of neurocognitive decline inpatients with MPS II (Hunter syndrome); and (b) reductions in biomarkersof disease, e.g., GAG levels and/or enzyme activity (IDS orhexosaminidase) in the CSF, serum and/or urine, and/or liver and spleenvolumes. Neurocognition in infants can be measured via Bayley Scales ofInfant and Toddler Development, Third Ed., BSID-III. Neurocognitive andadaptive behavioral assessments (e.g., using Bayley Scales of InfantDevelopment and Vineland Adaptive Behavior Scales, respectively) can beperformed.

Prior to treatment, the MPS II patient can be assessed for neutralizingantibodies (Nab) to the capsid of the rAAV vector used to deliver thehIDS gene. Such Nabs can interfere with transduction efficiency andreduce therapeutic efficacy. MPS II patients that have a baseline serumNab titer ≤1:5 are good candidates for treatment with the rAAV.hIDS genetherapy protocol. Treatment of MPS II patients with titers of serumNab>1:5 may require a combination therapy, such as transientco-treatment with an immunosuppressant before and/or during treatmentwith rAAV.hIDS vector delivery. Optionally, immunosuppressive co-therapymay be used as a precautionary measure without prior assessment ofneutralizing antibodies to the AAV vector capsid and/or other componentsof the formulation. In certain embodiments, prior immunosuppressiontherapy may be desirable to prevent potential adverse immune reaction tothe hIDS transgene product, especially in patients who have virtually nolevels of IDS activity, where the transgene product may be seen as“foreign.” While a reaction similar to that observed in animals may notoccur in human subjects, as a precaution immunosuppression therapy isrecommended for all recipients of rAAV-hIDS.

Combinations of gene therapy delivery of the rAAV.hIDS to the CNSaccompanied by systemic delivery of hIDS are encompassed by the methodsof the invention. Systemic delivery can be accomplished using ERTinfusions of idursulfase (e.g., using Elaprase®), or additional genetherapy using an rAAV.hIDS with tropism for the liver (e.g., anrAAV.hIDS bearing an AAV8 capsid).

In certain embodiments, the patient is administered an AAV.hIDS vialiver-directed injections in order to tolerize the patient to hIDS, andthe patient is subsequently administered AAV.hIDS via intrathecalinjections when the patient is an infant, child, and/or adult to expresstherapeutic concentrations of hIDS in the CNS.

Certain embodiments are based, in part, on (i) promising data generatedin a mouse model of MPS II (described in Example 2, infra) demonstratingthat treatment with the rAAV.hIDS vectors of the invention normalizedIDS expression; decreased biomarkers of disease; and improved behavioral(CNS) symptoms; and (ii) safety and biodistribution studies in nonhumanprimates (NHPs) as described in Example 3, infra. Certain embodiments ofthe invention are also illustrated by way of examples that describe themanufacture and characterization of the rAAV.hIDS pharmaceuticalcompositions (Examples 4 and 5, infra).

As used herein, the terms “intrathecal delivery” or “intrathecaladministration” refer to a route of administration for drugs via aninjection into the spinal canal, more specifically into the subarachnoidspace so that it reaches the cerebrospinal fluid (CSF). Intrathecaldelivery may include lumbar puncture, intraventricular,suboccipital/intracisternal, and/or C1-2 puncture. For example, materialmay be introduced for diffusion throughout the subarachnoid space bymeans of lumbar puncture. In another example, injection may be into thecisterna magna.

As used herein, the terms “intracisternal delivery” or “intracisternaladministration” refer to a route of administration for drugs directlyinto the cerebrospinal fluid of the cisterna magna cerebellomedularis,more specifically via a suboccipital puncture or by direct injectioninto the cisterna magna or via permanently positioned tube.

As used herein, a “therapeutically effective amount” refers to theamount of the AAV.hIDS composition which delivers and expresses in thetarget cells an amount of enzyme sufficient to ameliorate or treat oneor more of the symptoms of MPS II. “Treatment” may include preventingthe worsening of the symptoms of one of the MPS II syndromes andpossibly reversal of one or more of the symptoms thereof. For example, atherapeutically effective amount of rAAV.hIDS is the amount whichimproves neurocognitive function in a patient having MPS II. Improvementof such neurocognitive function may be measured by assessing subjects'neurocognitive developmental quotient (DQ), using Bayley Scales ofInfant and Toddler Development. Improvement of neurocognitive functionmay also be measured by assessing subjects' intelligence quotient (IQ),using methods known in the art including but not limited to, e.g., useof the Wechsler Abbreviated Scale of Intelligence (WASI) (IQ), Bayley'sInfantile Development Scale, the Hopkins Verbal Learning Test (memory),and/or the Tests of Variables of Attention (TOVA). In anotherembodiment, a therapeutically effective amount of rAAV.hIDS is theamount which decreases pathogenic GAG, heparan sulfate, and/orhexosaminidase concentration in urine and/or cerebrospinal fluid and/orserum and/or other tissues. In still other embodiments, correction ofcorneal clouding may be observed, correction of lesions in the centralnervous system (CNS) is observed, and/or reversal of perivascular and/ormeningeal gag storage is observed.

A “therapeutically effective amount” may be determined based on ananimal model, rather than a human patient. Examples of a suitable murinemodel are described herein.

As used herein a “functional human iduronate-2-sulfatase” refers to ahuman iduronate-2-sulfatase enzyme which functions normally in humanswithout MPS II or an associated syndrome. Conversely, a humaniduronate-2-sulfatase enzyme variant which causes MPS II or anassociated syndrome is considered non-functional. In one embodiment, afunctional human iduronate-2-sulfatase has the amino acid sequence of awild-type human iduronate-2-sulfatase described by Wilson et al, Proc.Natl. Acad. Sci. U.S.A. 87 (21): 8531-8535 (1990), NCBI ReferenceSequence NP_000193.1, reproduced in SEQ ID NO: 2 (550 amino acids); thispreproprotein includes a signal peptide (amino acids 1 to 25), apro-peptide (amino acids 26 to 33) and a mature peptide composed ofamino acids 34 to 455 (a 42 kDa chain) and amino acids 456 to 550 (a 14kDa chain). See, also, UniProtKB/Swiss-Prot (P22304.1)

As used herein, the term “NAb titer” refers to a measurement of how muchneutralizing antibody (e.g., anti-AAV Nab) is produced which neutralizesthe physiologic effect of its targeted epitope (e.g., an AAV). Anti-AAVNAb titers may be measured as described in, e.g., Calcedo, R., et al.,Worldwide Epidemiology of Neutralizing Antibodies to Adeno-AssociatedViruses. Journal of Infectious Diseases, 2009. 199(3): p. 381-390, whichis incorporated by reference herein.

As used herein, an “expression cassette” refers to a nucleic acidmolecule which comprises an IDS gene, promoter, and may include otherregulatory sequences therefor, which cassette may be delivered via agenetic element (e.g., a plasmid) to a packaging host cell and packagedinto the capsid of a viral vector (e.g., a viral particle). Typically,such an expression cassette for generating a viral vector contains theIDS coding sequence described herein flanked by packaging signals of theviral genome and other expression control sequences such as thosedescribed herein.

The abbreviation “sc” refers to self-complementary. “Self-complementaryAAV” refers a construct in which a coding region carried by arecombinant AAV nucleic acid sequence has been designed to form anintra-molecular double-stranded DNA template. Upon infection, ratherthan waiting for cell mediated synthesis of the second strand, the twocomplementary halves of scAAV will associate to form one double strandedDNA (dsDNA) unit that is ready for immediate replication andtranscription. See, e.g., D M McCarty et al, “Self-complementaryrecombinant adeno-associated virus (scAAV) vectors promote efficienttransduction independently of DNA synthesis”, Gene Therapy, (August2001), Vol 8, Number 16, Pages 1248-1254. Self-complementary AAVs aredescribed in, e.g., U.S. Pat. Nos. 6,596,535; 7,125,717; and 7,456,683,each of which is incorporated herein by reference in its entirety.

As used herein, the term “operably linked” refers to both expressioncontrol sequences that are contiguous with the gene of interest andexpression control sequences that act in trans or at a distance tocontrol the gene of interest.

The term “heterologous” when used with reference to a protein or anucleic acid indicates that the protein or the nucleic acid comprisestwo or more sequences or subsequences which are not found in the samerelationship to each other in nature. For instance, the nucleic acid istypically recombinantly produced, having two or more sequences fromunrelated genes arranged to make a new functional nucleic acid. Forexample, in one embodiment, the nucleic acid has a promoter from onegene arranged to direct the expression of a coding sequence from adifferent gene. Thus, with reference to the coding sequence, thepromoter is heterologous.

A “replication-defective virus” or “viral vector” refers to a syntheticor artificial viral particle in which an expression cassette containinga gene of interest is packaged in a viral capsid or envelope, where anyviral genomic sequences also packaged within the viral capsid orenvelope are replication-deficient; i.e., they cannot generate progenyvirions but retain the ability to infect target cells. In oneembodiment, the genome of the viral vector does not include genesencoding the enzymes required to replicate (the genome can be engineeredto be “gutless”—containing only the transgene of interest flanked by thesignals required for amplification and packaging of the artificialgenome), but these genes may be supplied during production. Therefore,it is deemed safe for use in gene therapy since replication andinfection by progeny virions cannot occur except in the presence of theviral enzyme required for replication.

As used herein, “recombinant AAV9 viral particle” refers tonuclease-resistant particle (NRP) which has an AAV9 capsid, the capsidhaving packaged therein a heterologous nucleic acid molecule comprisingan expression cassette for a desired gene product. Such an expressioncassette typically contains an AAV 5′ and/or 3′ inverted terminal repeatsequence flanking a gene sequence, in which the gene sequence isoperably linked to expression control sequences. These and othersuitable elements of the expression cassette are described in moredetail below and may alternatively be referred to herein as thetransgene genomic sequences. This may also be referred to as a “full”AAV capsid. Such a rAAV viral particle is termed “pharmacologicallyactive” when it delivers the transgene to a host cell which is capableof expressing the desired gene product carried by the expressioncassette.

In many instances, rAAV particles are referred to as “DNase resistant.”However, in addition to this endonuclease (DNase), other endo- andexo-nucleases may also be used in the purification steps describedherein, to remove contaminating nucleic acids. Such nucleases may beselected to degrade single stranded DNA and/or double-stranded DNA, andRNA. Such steps may contain a single nuclease, or mixtures of nucleasesdirected to different targets, and may be endonucleases or exonucleases.

The term “nuclease-resistant” indicates that the AAV capsid has fullyassembled around the expression cassette which is designed to deliver atransgene to a host cell and protects these packaged genomic sequencesfrom degradation (digestion) during nuclease incubation steps designedto remove contaminating nucleic acids which may be present from theproduction process.

As used herein, “AAV9 capsid” refers to the AAV9 having the amino acidsequence of GenBank accession: AAS99264, is incorporated by referenceherein and the AAV vp1 capsid protein is reproduced in SEQ ID NO: 13.Some variation from this encoded sequence is encompassed by the presentinvention, which may include sequences having about 99% identity to thereferenced amino acid sequence in GenBank accession: AAS99264, SEQ IDNO: 13 and U.S. Pat. No. 7,906,111 (also WO 2005/033321) (i.e., lessthan about 1% variation from the referenced sequence). Such AAV mayinclude, e.g., natural isolates (e.g., hu31 or hu32), or variants ofAAV9 having amino acid substitutions, deletions or additions, e.g.,including but not limited to amino acid substitutions selected fromalternate residues “recruited” from the corresponding position in anyother AAV capsid aligned with the AAV9 capsid; e.g., such as describedin U.S. Pat. Nos. 9,102,949, 8,927,514, US2015/349911; and WO2016/049230A1. However, in other embodiments, other variants of AAV9, orAAV9 capsids having at least about 95% identity to the above-referencedsequences may be selected. See, e.g., US Published Patent ApplicationNo. 2015/0079038. Methods of generating the capsid, coding sequencestherefore, and methods for production of rAAV viral vectors have beendescribed. See, e.g., Gao, et al, Proc. Natl. Acad. Sci. U.S.A. 100(10), 6081-6086 (2003) and US 2013/0045186A1.

The term “AAV9 intermediate” or “AAV9 vector intermediate” refers to anassembled rAAV capsid which lacks the desired genomic sequences packagedtherein. These may also be termed an “empty” capsid. Such a capsid maycontain no detectable genomic sequences of an expression cassette, oronly partially packaged genomic sequences which are insufficient toachieve expression of the gene product. These empty capsids arenon-functional to transfer the gene of interest to a host cell.

The term “a” or “an” refers to one or more. As such, the terms “a” (or“an”), “one or more,” and “at least one” are used interchangeablyherein.

The words “comprise”, “comprises”, and “comprising” are to beinterpreted inclusively rather than exclusively. The words “consist”,“consisting”, and its variants, are to be interpreted exclusively,rather than inclusively. While various embodiments in the specificationare presented using “comprising” language, under other circumstances, arelated embodiment is also intended to be interpreted and describedusing “consisting of” or “consisting essentially of” language.

The term “about” encompasses a variation within and including ±10%,unless otherwise specified.

Unless defined otherwise in this specification, technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art and by reference to published texts, whichprovide one skilled in the art with a general guide to many of the termsused in the present application.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the AAV9.CB.hIDS vector genome.The IDS expression cassette is flanked by inverted terminal repeats(ITRs) and expression is driven by a hybrid of the cytomegalovirus (CMV)enhancer and the chicken beta actin promoter (CB7). The transgeneincludes the chicken beta actin intron and a rabbit beta-globinpolyadenylation (polyA) signal.

FIGS. 2A-2C provides IDS expression in CNS and serum of MPS II miceafter IT vector treatment. MPS II mice were treated at 2-3 months of agewith an ICV injection of AAV9.CB.hIDS at one of three doses: 3×10⁸ GC(low), 3×10⁹ GC (mid) or 3×10¹⁰ GC (high). Animals were sacrificed threeweeks after injection. IDS activity was measured in CSF (FIG. 2A) wholebrain homogenate (FIG. 2B) and serum (FIG. 2C). Wild type and untreatedMPS II mice were used as controls.

FIG. 3 provides biodistribution of vector DNA in MPS II miceadministered AAV9.CB.hIDS. MPS II mice were treated with an ICVinjection of 3×10¹⁰ GC of an AAV9 vector. Three weeks after injection,the animals were sacrificed and vector genomes were quantified in tissueDNA by Taqman PCR.

FIGS. 4A-4D provide correction of peripheral GAG after IT vectordelivery in MPS II Mice. MPS II mice were treated at 2-3 months of agewith an ICV injection of AAV9.CB.hIDS at one of three doses: 3×10⁸ GC(low), 3×10⁹ GC (mid), or 3×10¹⁰ GC (high). Animals were sacrificedthree months after injection. Hexosaminidase activity was measured inliver (FIG. 4A) and heart (FIG. 4B). Storage correction was seen at alldoses in the liver and at mid- and high-doses in the heart. GAG contentwas measured in liver (FIG. 4C) and heart (FIG. 4D). Wild type anduntreated MPS II mice were used as controls. *p<0.05, one way ANOVAfollowed by Dunnett's test.

FIGS. 5A-5L provide dose-dependent resolution of brain storage lesionsin MPS II Mice. MPS II mice were treated at 2-3 months of age with anICV injection of AAV9.CB.hIDS at one of three doses: 3×10⁸ GC (low),3×10⁹ GC (mid), or 3×10¹⁰ GC (high) Animals were sacrificed three monthsafter injection, and brains were stained for the lysosomal membraneprotein LIMP2 and the ganglioside GM3. Cells staining positive for GM3(FIGS. 5A, 5C, 5 E, 5G and 5I) and LIMP2 (5B, 5D, 5F, 5H and 5J) werequantified by a blinded reviewer in four cortical brain sections fromeach animal Representative cortical brain sections are shown (GM3, FIG.5K; LIMP2, FIG. 5L). Wild type and untreated MPS II mice were used ascontrols. *p<0.05, one way ANOVA followed by Dunnett's test.

FIGS. 6A-6C shows improved object discrimination in vector-treated MPSII Mice. Untreated MPS II mice and WT male littermates underwentbehavioral testing at 4-5 months of age. MPS II mice were treated at 2-3months of age with an ICV injection of AAV9.CB.hIDS at one of threedoses: 3×10⁸ GC (low), 3×10⁹ GC (mid) or 3×10¹⁰ GC (high). Two monthsafter injection animals underwent behavioral testing. FIG. 6Aillustrates Y maze behavior and FIG. 6B illustrates contextual fearconditioning, which were evaluated 2 months after injection by a blindedreviewer. “Pre” indicated freezing time when reexposed to the enclosure24 hours after receiving an un-signaled 1.5 mA foot shock. *p<0.05,two-way ANOVA followed by Sidak's multiple comparisons test. FIG. 6Cshows the percentage time spent exploring a novel or familiar object inthe novel object recognition task, which is used to assess long termmemory. Wild type and untreated MPS II mice were used as controls.*p<0.05, t-test with Bonferroni correction for multiple comparisons.

FIGS. 7A-7B provide a comparison of enzyme expression and correction ofbrain storage lesions in MPS I mice treated with IT AAV9. MPS I micewere treated at 2-3 months of age with an ICV injection of AAV9.CB.hIDUAat one of three doses: 3×10⁸ GC (low), 3×10⁹ GC (mid), or 3×10¹⁰ GC(high). One cohort of animals was sacrificed at 3 weeks post vectorinjection, and brains were harvested for measurement of IDUA activity.This is shown in FIG. 7A. FIG. 7B shows a second cohort of animals wassacrificed 3 months after injection, and brains were stained for thelysosomal membrane protein LIMP2. Cells staining positive for LIMP2 werequantified by a blinded reviewer in 4 cortical brain sections. Wild typeand untreated MPS II mice were used as controls. *p<0.05, one-way ANOVAfollowed by Dunnett's test.

FIG. 8 illustrates the antibody response against human IDS in MPS IImice treated with ICV AAV9.CB.hIDS. MPS II mice were treated at 2-3months of age with an ICV injection of AAV9.CB.hIDS at one of threedoses: 3×10⁸ GC (low), 3×10⁹ GC (mid), or 3×10¹⁰ GC (high). Serum wascollected at necropsy from mice sacrificed at day 21 or day 90 postvector administration. Antibodies to human IDS were evaluated byindirect ELISA. Dashed line indicates 2 SD above the mean titer in naïveserum from untreated animals. Most animals had no detectable antibodies(in the background level of naïve sera).

FIGS. 9A-9D illustrate the normal open field activity and Y mazeperformance in MPS II mice. Untreated MPS II mice and WT malelittermates underwent behavioral testing at 4-5 months of age. Openfield activity was measured by XY axis beam breaks for horizontalactivity (FIG. 9A), Z axis beam breaks for vertical activity (FIG. 9B)and percent center beam breaks for center activity (FIG. 9C). Total armentries (FIG. 9D) were recorded during an 8 min Y maze testing session

FIGS. 10A-10B provides a manufacturing process flow diagram.

FIG. 11 is an image of apparatus (10) for intracisternal delivery of apharmaceutical composition, including optional introducer needle forcoaxial insertion method (28), which includes a 10 cc vector syringe(12), a 10 cc prefilled flush syringe (14), a T-connector extension set(including tubing (20), a clip at the end of the tubing (22) andconnector (24)), a 22 G×5″ spinal needle (26), and an optional 18 G×3.5″introducer needle (28). Also illustrated is the 4-way stopcock withswive male luer lock (16).

FIGS. 12A-12I illustrate encephalitis and transgene specific T cellresponses in dogs treated with ICV AAV9. One-year-old MPS I dogs weretreated with a single ICV or IC injection of an AAV9 vector expressingGFP. All animals were sacrificed 14 days after injection, except for1-567 which was found dead 12 days after injection. Brains were dividedinto coronal sections, which revealed gross lesions near the injectionsite (arrowheads) in ICV treated animals (FIGS. 12A to 12F). Tissuesections from the brain regions surrounding the gross lesions werestained with hematoxylin and eosin (FIGS. 12G and 12H). Originalmagnification=4× (left panel), and 20× (right panel). Peripheral bloodmononuclear cells were collected from one ICV treated dog (I-565) at thetime of necropsy, and T cell responses against the AAV9 capsid and humanIDUA protein were measured by interferon-γ ELISPOT (FIG. 12I). T cellresponses to the GFP transgene product were measured using a single poolof overlapping 15 amino acid peptides covering the full GFP sequence.The peptides comprising the AAV9 capsid protein were divided into threepools (designated pool A-C). *=positive response, defined as >3-foldbackground (unstimulated cells) and greater than 55 spots per millioncells. Phytohaemagglutinin (PHA) and ionomycin with phorbol 12-myristate13-acetate (PMA) served as positive controls for T cell activation.

FIG. 13 is a bar chart illustrating vector biodistribution in dogstreated with ICV or IC AAV9. Dogs were sacrificed 14 days afterinjection with a single ICV or IC injection of an AAV9 vector expressingGFP, except for animal I-567 which was necropsied 12 days afterinjection. Vector genomes were detected in tissue samples byquantitative PCR. Values are expressed as vector genome copies perdiploid cell (GC/diploid genome). Brain samples collected from thehippocampus or cerebral cortex are indicated as either injected oruninjected hemisphere for the ICV treated dogs; for the IC treatedanimals these are the right and left hemispheres, respectively. Sampleswere not collected for PCR from the injected cerebral hemisphere ofanimal I-567.

FIGS. 14A to 14H show GFP expression in brain and spinal cord of dogstreated with ICV or IC AAV9. GFP expression was evaluated by directfluorescence microscopy of brain and spinal cord samples collected fromdogs treated with ICV or IC injection of an AAV9 vector expressing GFP.Representative sections are shown for samples of frontal cortex, andfrom the anterior horn of the spinal cord collected at the cervical,thoracic, and lumbar levels. Original magnification=10×.

FIGS. 15A-15Q show stable transgene expression and absence ofencephalitis in an MPS VII dog treated with ICV AAV9 expressing thelysosomal enzyme β-glucuronidase (GUSB). A 6-week-old dog with geneticdeficiency of GUSB (a model of MPS VII) was treated with a single ICVinjection of an AAV9 vector expressing GUSB. GUSB enzyme activity wasmeasured in CSF samples collected at the time of injection and on day 7and 21 after injection (FIG. 15A). The dog was sacrificed three weeksafter injection. Gross and microscopic evaluation of the brain regionssurrounding the injection site was performed (FIGS. 15B to 15D).Original magnification=4× (center panel), and 10× (right panel). GUSBactivity was detected in brain and spinal cord sections using asubstrate which produces a red product when cleaved by active GUSB(FIGS. 15E-15N). Representative sections are shown for samples ofcerebral cortex, cerebellum, and the anterior horn of the spinal cordcollected at the cervical, thoracic, and lumbar levels. Originalmagnification=4× (cortex and cerebellum), and 10× (spinal cord).Sections of cerebral cortex collected from an untreated MPS VII dog, anormal dog, and the MPS VII dog treated with ICV AAV9 were stained forthe ganglioside GM3, which pathologically accumulates in the brains ofMPS VII dogs (FIGS. 15O to 15Q). Original magnification=4×.

FIGS. 16A to 16B show contrast distribution after lumbar intrathecalinjection in non human primates (NHPs). Adult cynomolgus macaquesreceived an intrathecal injection via lumbar puncture of an AAV9 vectordiluted in 5 mL of Iohexol 180. The distribution of contrast along thespinal cord was evaluated by fluoroscopy. Representative images of thethoracic and cervical regions are shown. Contrast material (arrowheads)was visible along the entire length of the spinal cord within 10 minutesof injection in all animals.

FIG. 17 is a bar chart showing vector biodistribution in NHPs treatedwith intrathecal AAV9. NHPs were sacrificed 14 days after intrathecalinjection via lumbar puncture of an AAV9 vector diluted in 5 mL ofIohexol 180. Two of the animals were placed in the Trendelenburgposition for 10 minutes after injection. Vector genomes were detected intissue samples by quantitative PCR. Values are expressed as vectorgenome copies per diploid cell (GC/diploid genome).

FIGS. 18A to 18H show GFP expression in brain and spinal cord of NHPstreated with intrathecal AAV9. GFP expression was evaluated by directfluorescence microscopy of brain and spinal cord samples collected fromNHPs treated with intrathecal injection of an AAV9 vector. The vectorwas administered by lumbar puncture. Two of the animals were placed inthe Trendelenburg position for 10 minutes after injection.Representative sections are shown for samples of frontal cortex, andfrom the anterior horn of the spinal cord collected at the cervical,thoracic, and lumbar levels. Due to the presence of autofluorescentmaterial in some NHP tissues, red channel images were captured todifferentiate autofluorescence from GFP signal. Autofluorescence imagesare overlaid in magenta. Original magnification=4× (cortex), and 10×(spinal cord).

FIGS. 19A-19B illustrate elevated CSF spermine in MPS I. A highthroughput LC/MS and GC/MS metabolite screen was performed on CSFsamples from MPS I dogs (n=15) and normal controls (n=15). A heatmap ofthe top 100 differentially detected metabolites (ANOVA) is shown (FIG.19A). The youngest animal in the MPS I cohort (28 days of age) isindicated by an asterisk. Spermine concentration was measured by aquantitative isotope dilution LC/MS assay in CSF samples from 6 infantswith MPS I and 2 normal infants (FIG. 19B).

FIGS. 20A-20J illustrate spermine dependent aberrant neurite growth inMPS I neurons. Cortical neurons harvested from E18 wild-type or MPS Imouse embryos were treated with spermine (50 ng/mL) or the sperminesynthase inhibitor APCHA 24 hours after plating. Phase contrast imageswere acquired 96 hours after plating (FIGS. 20A to 20D). Neurite number,length and branching were quantified for 45-65 randomly selected neuronsfrom duplicate cultures per treatment condition by a blinded reviewer(FIGS. 20E&20H, 20G&20J, and 20F&201). *** p<0.0001 (ANOVA followed byDunnett's test).

FIGS. 21A-21K illustrate normalization of CSF spermine levels and brainGAP43 expression in MPS I dogs following gene therapy. Five MPS I dogswere treated with an intrathecal injection of an AAV9 vector expressingcanine IDUA at one month of age. Two of the dogs (I-549, I-552) weretolerized to IDUA by liver directed gene therapy on postnatal day 1 inorder to prevent the antibody response that is elicited to IDUA in someMPS I dogs. Six months after intrathecal vector injection, IDUA activitywas measured in brain tissue (FIG. 21A). Brain storage lesions wereassessed by staining for the lysosomal membrane protein LIMP2 (FIGS. 21Bto 21H). GAP43 was measured in cortical brain samples by western blot(FIG. 21I) and quantified relative to β-actin by densitometry (FIG.21J). CSF spermine was measured at the time of sacrifice by isotopedilution LC/MS (FIG. 21K). Untreated MPS I dogs (n=3) and normal dogs(n=2) served as controls. * p<0.05 (Kruskal-Wallis test followed byDunn's test).

FIGS. 22A-22B illustrate the use of spermine as a CSF biomarker forevaluation of CNS directed gene therapy in MPS I. Six MPS I dogstolerized to human IDUA at birth were treated with intrathecal AAV9expressing human IDUA (10¹² GC/kg, n=2, 10¹¹ GC/kg, n=2, 10¹⁰ GC/kg,n=2) at one month of age. CSF spermine levels were measured six monthsafter treatment (FIG. 22A). Three MPS I cats were treated withintrathecal AAV9 expressing feline IDUA (10¹² GC/kg). CSF spermine wasquantified six months after treatment (FIG. 22B). Untreated MPS I dogs(n=3) and normal dogs (n=2) served as controls.

FIG. 23 illustrates the mean decrease accuracy for metabolitesidentified by random forest analysis.

FIG. 24 illustrates the expression of enzymes in the polyamine syntheticpathway in MPS I dog brain samples.

FIG. 25 illustrates spermine concentration in MPS VII dog CSF

FIGS. 26A to 26C illustrates that there is no impact of APCHA treatmenton WT neuron growth.

FIG. 27 illustrates dose dependent reduction of MPS II-related pathologyfindings in MPS II mice (IDS^(γ/−)) treated with ICVAAV9.CB7.CI.hIDS.rBG with various doses (3e8, 3×10⁸ GC; 3e9, 3×10⁹ GC;3e10, 3×10¹⁰ GC). Cumulative Pathology Scores were evaluated and plottedon the y-axis. Heterozygous mice (IDS^(γ/+)) served as control. Resultsdemonstrated a dose dependent reduction of MPS II-related pathologyfindings in MPS II mice treated with ICV AAV9.CB7.CI.hIDS.rBG.

FIG. 28 illustrates CSF pleocytosis in the Rhesus Macaques described inExample 9. White blood cells (leukocytes, y axis) were counted in theCSF samples collected at various days (x axis, Day 0, Day 7, Day 14, Day21, Day 30, Day 45, Day 60 and Day 90). Diamonds represent data from RA2198 without treatment with AAV9.CB7.hIDS. Squares, triangles, and xrepresent data from RA 1399, RA 2203 and RA 2231 treated with 5×10¹³ GCof AAV9.CB7.hIDS respectively. *, circles and + represent data from RA1358, RA 1356 and RA 2197 treated with 1.7×10¹³ GC of AAV9.CB7.hIDSrespectively.

FIG. 29 shows ELISA results measuring anti hIDS antibody developed inthe serum of NHP on Day 60 as described in Example 9. Dilution wasplotted as x axis while optical density (OD) as y axis. Diamondsrepresent data from RA 2198 without treatment with AAV9.CB7.hIDS.Squares represent data from RA 2197 treated with 1.7×10¹³ GC ofAAV9.CB7.hIDS while triangles and x represent that of RA 2203 and RA2231 treated with 5×10¹³ GC of AAV9.CB7.hIDS respectively.

5. DETAILED DESCRIPTION OF THE INVENTION

The use of a replication deficient AAV to deliver a hIDS gene to the CNSof patients (human subjects) diagnosed with MPS II is provided. Therecombinant AAV (“rAAV”) vector used for delivering the hIDS gene(“rAAV.hIDS”) has tropism for the CNS (e.g., an rAAV bearing an AAV9capsid), and the hIDS transgene is controlled by specific expressioncontrol elements, e.g., a hybrid of cytomegalovirus (CMV) enhancer andthe chicken beta actin promoter (CB7). In certain embodiments,pharmaceutical compositions suitable for intrathecal, intracisternal,and systemic administration, which comprise a suspension of rAAV.hIDSvectors in a formulation buffer comprising a physiologically compatibleaqueous buffer, a surfactant and optional excipients are provided. TherAAV suspension is further characterized in that:

(i) the rAAV Genome Copy (GC) titer is at least 1.0×10¹³ GC/mL;

(ii) the rAAV Empty/Full particle ratio is between 0.01 and 0.05(95%-99% free of empty capsids) as determined by SDS-PAGE analysis (seeExample 5D); in other embodiments, the rAAV9.hIDS provided herein are atleast about 80%, at least about 85%, or at least about 90% free of emptycapsids; and/or

(iii) a dose of at least about 4×10⁸ GC/g brain mass to about 4×10¹¹GC/g brain mass of the rAAV suspension has potency.

Potency can be measured by in vitro cell culture assays, e.g., the invitro potency assay described in Example 5G, in which HEK293 cells aretransduced with a known multiplicity of rAAV GCs per cell and thesupernatant is assayed for IDS activity 72 hours post-transduction. Thefunction (activity) and/or the potency of hIDS may be measured in asuitable in vitro assay, e.g., using the 4MU-iduronide enzymatic assaywhich measures the ability of hIDS to cleave a fluorogenic substrate,4-Methylumbelliferyl alpha-L-iduronide-2 sulfate. The specific activityis >7,500 pmol/min/μg, as measured under the described conditions. SeeActivity Assay Protocol on www.RnDSystems.com. Other suitable methods ofmeasuring enzyme activity have been described [see, e.g., Kakkis, E. D.,et al (1994). Protein Expression Purif. 5: 225-232; Rome, L. H., et al(1979). Proc. Natl. Acad. Sci. USA 76: 2331-2334], including thosedescribed herein. Activity may also be assessed using the methoddescribed, e.g., E. Oussoren, et al, Mol Genet Metab. 2013 August;109(4):377-81. doi: 10.1016/j.ymgme.2013.05.016. Epub 2013 Jun. 4.Patients who are candidates for treatment are pediatric and adultpatients with MPS II (Hunter syndrome) and/or the symptoms associatedwith MPS II.

The following therapeutically effective flat doses of rAAV9.hIDS can beadministered to MPS II patients of the indicated age group:

-   -   Newborns: about 3.8×10¹² to about 1.9×10¹⁴ GC;    -   3-9 months: about 6×10¹² to about 3×10¹⁴ GC;    -   9-36 months: about 10¹³ to about 5×10¹⁴ GC;    -   3-12 years: about 1.2×10¹³ to about 6×10¹⁴ GC;    -   12+ years: about 1.4×10¹³ to about 7.0×10¹⁴ GC;    -   18+ years (adult): about 1.4×10¹³ to about 7.0×10¹⁴ GC.

In some embodiments, the dose administered to a 12+ year old MPIIpatient (including 18+ year old) is 1.4×10¹³ genome copies (GC)(1.1×10¹⁰ GC/g brain mass). In some embodiments, the dose administeredto a 12+ year old MPII patient (including 18+ year old) is 7×10¹³ GC(5.6×10¹⁰ GC/g brain mass). In still a further embodiment, the doseadministered to an MPSII patient is at least about 4×10⁸ GC/g brain massto about 4×10¹¹ GC/g brain mass. In certain embodiments, the doseadministered to MPS II newborns ranges from about 1.4×10¹¹ to about1.4×10¹⁴ GC; the dose administered to infants 3-9 months ranges fromabout 2.4×10¹¹ to about 2.4×10¹⁴ GC; the dose administered to MPS IIchildren 9-36 months ranges: about 4×10¹¹ to about 4×10¹⁴ GC; the doseadministered to MPS II children 3-12 years: ranges from about 4.8×10¹¹to about 4.8×10¹⁴ GC; the dose administered to children and adults 12+years ranges from about 5.6×10¹¹ to about 5.6×10¹⁴ GC.

The goal of the treatment is to functionally replace the patient'sdefective iduronate-2-sulfatase via rAAV-based CNS-directed gene therapyas a viable approach to treat disease. As expressed from the rAAV vectordescribed herein, expression levels of at least about 2% as detected inthe CSF, serum, neurons, or other tissue, may provide therapeuticeffect. However, higher expression levels may be achieved. Suchexpression levels may be from 2% to about 100% of normal functionalhuman IDS levels. In certain embodiments, higher than normal expressionlevels may be detected in CSF, serum, or other tissue.

Efficacy of the therapy can be measured by assessing (a) the preventionof neurocognitive decline in patients with MPS II (Hunter syndrome); and(b) reductions in biomarkers of disease, e.g., GAG levels and/or enzymeactivity in the CSF, serum and/or urine, and/or liver and spleenvolumes. Neurocognition in infants can be measured via Bayley Scales ofInfant and Toddler Development, Third Ed., BSID-III. Neurocognitive andadaptive behavioral assessments (e.g., using Bayley Scales of InfantDevelopment and Vineland Adaptive Behavior Scales, respectively) can beperformed.

Combinations of gene therapy delivery of the rAAV.hIDS to the CNSaccompanied by systemic delivery of hIDS are encompassed by certainembodiments. Systemic delivery can be accomplished using ERT (e.g.,using Elaprase®), or additional gene therapy using an rAAV.hIDS withtropism for the liver (e.g., an rAAV.hIDS bearing an AAV8 capsid).

Certain embodiments also provide for the manufacture andcharacterization of the rAAV.hIDS pharmaceutical compositions (Example4, infra).

5.1. AAV.hIDS Constructs and Formulations

5.1.1. Expression Cassettes

An AAV vector that comprises an expression cassette containing a hIDSgene characterized by having the nucleotide sequence of SEQ ID NO: 1(CCDS 14685.1) is provided. This sequence is the published gene sequenceencoding Genbank NP000193.1, also enclosed herein as SEQ ID NO: 2. Inanother embodiment, the expression cassette contains a hIDS genecharacterized by having the nucleotide sequence at least about 75%identical to SEQ ID NO: 1 and encodes a functional humaniduronate-2-sulfatase. In another embodiment, the expression cassettecontains a hIDS gene characterized by having the nucleotide sequence atleast about 80% identical to SEQ ID NO: 1 and encodes a functional humaniduronate-2-sulfatase. In another embodiment, the sequence is at leastabout 85% identity to SEQ ID NO: 1 or at least about 90% identical toSEQ ID NO: 1 and encodes a functional human iduronate-2-sulfatase. Inone embodiment, the sequence is at least about 95% identical to SEQ IDNO: 1, at least about 97% identical to SEQ ID NO: 1, or at least about99% identical to SEQ ID NO: 1 and encodes a functional humaniduronate-2-sulfatase. In one embodiment, the sequence is at least about77% identical to SEQ ID NO: 1. In another embodiment, the expressioncassette contains a hIDS coding sequence of nt 1177 to nt 2829 of SEQ IDNO: 8, also shown as nt 1937 to nt 3589 of SEQ ID NO: 11. In anotherembodiment, the expression cassette contains a hIDS coding sequence atleast about 75%, about 80%, about 85%, about 90%, about 95%, about 97%,about 98% or about 99% identical to nt 1177 to nt 2829 of SEQ ID NO: 8(also shown as nt 1937 to nt 3589 of SEQ ID NO: 11).

In another embodiment, a functional human iduronate-2-sulfatase mayinclude a synthetic amino acid sequence in which all or a portion of thefirst 25 amino acids of the preproprotein SEQ ID NO: 2, which correspondto the leader (signal) peptide, are replaced with a heterologous leaderpeptide. This leader peptide, e.g., such as the leader peptides frominterleukin-2 (IL-2) or oncostatin, can improve transport of the enzymeout of the cell through its secretory pathway into the circulation.Suitable leader peptides are preferably, although not necessarily ofhuman original. Suitable leader peptides may be chosen fromproline.bic.nus.edu.sg/spdb/zhang270.htm, which is incorporated byreference herein, or may be determined using a variety of computationalprograms for determining the leader (signal) peptide in a selectedprotein. Although not limited, such sequences may be from about 15 toabout 50 amino acids in length, or about 19 to about 28 amino acids inlength, or may be larger or smaller as required. In addition, at leastone in vitro assay has been described as being useful to assess theenzymatic activity of an IDS enzyme [see, e.g., Dean et al, ClinicalChemistry, 2006 April; 52(4): 643-649]. In addition to removal of all ora portion of the preproprotein (amino acids 1 to 25 of SEQ ID NO: 2),all or a portion of the proprotein (amino acids 26 to 33 of SEQ ID NO:2) may be removed and optionally replaced with a heterologous maturepeptide.

In another embodiment, the AAV vector that comprises an expressioncassette further containing a SUMF1 gene characterized by having thenucleotide sequence of nt 3423 to nt 4547 of SEQ ID NO: 5 (CDS ofGenBank: AB448737.1) is provided. This sequence is the published genesequence encoding NCBI Reference Sequence: NP_877437.2, which is alsoenclosed herein as SEQ ID NO: 7 and SEQ ID NO: 10. Some studies havesuggested that expression of sulfatases such as IDS can be limited bythe availability of the sulfatase modifying factor, SUMF1, which isrequired for post-translational modification of IDS (Fraldi, et al,Biochemical J, 2007: 403: 305-312). In another embodiment, theexpression cassette contains a SUMF1 gene characterized by having thenucleotide sequence at least about 80% identical to nt 3423 to nt 4547of SEQ ID NO: 5 and encodes a functional human SUMF1. In anotherembodiment, the sequence is at least about 85% identity to nt 3423 to nt4547 of SEQ ID NO: 5 or at least about 90% identical to nt 3423 to nt4547 of SEQ ID NO: 5 and encodes a functional human SUMF1. In oneembodiment, the sequence is at least about 95%, about 97%, or about 99%identical to nt 3423 to nt 4547 of SEQ ID NO: 5 and encodes a functionalhuman SUMF1. In one embodiment, the sequence is at least about 76.6%identical to nt 3423 to nt 4547 of SEQ ID NO: 5. In another embodiment,the expression cassette contains a hSUMF1 coding sequence of nt 3423 tont 4553 of SEQ ID NO: 8. In another embodiment, the expression cassettecontains a hSUMF1 coding sequence at least about 75%, about 80%, about85%, about 90%, about 95%, about 97%, about 98% or about 99% identicalto nt 3423 to nt 4553 of SEQ ID NO: 8.

In one embodiment, an AAV vector that comprises an expression cassettecontaining a hIDS encoding sequence and a hSUMF1 encoding sequence isprovided. In another embodiment, the hIDS encoding sequence and hSUMF1encoding sequence were linked by an internal ribosome entry site (IRES).In a further embodiment, the IRES has a sequence of nt 2830 to nt 3422of SEQ ID NO: 5 or nt 2830 to nt 3422 of SEQ ID NO: 8. In anotherembodiment, the expression cassette contains nt 1177 to nt 4547 of SEQID NO: 5 or nt 1177 to nt 4553 of SEQ ID NO: 8 comprising an hIDSencoding sequence, an IRES and an hSUMF1 encoding sequence.

Identity or similarity with respect to a sequence is defined herein asthe percentage of amino acid residues in the candidate sequence that areidentical (i.e., same residue) or similar (i.e., amino acid residue fromthe same group based on common side-chain properties, see below) withthe peptide and polypeptide regions provided herein, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. Percent (%) identity is a measure of therelationship between two polynucleotides or two polypeptides, asdetermined by comparing their nucleotide or amino acid sequences,respectively. In general, the two sequences to be compared are alignedto give a maximum correlation between the sequences. The alignment ofthe two sequences is examined and the number of positions giving anexact amino acid or nucleotide correspondence between the two sequencesdetermined, divided by the total length of the alignment and multipliedby 100 to give a % identity figure. This % identity figure may bedetermined over the whole length of the sequences to be compared, whichis particularly suitable for sequences of the same or very similarlength and which are highly homologous, or over shorter defined lengths,which is more suitable for sequences of unequal length or which have alower level of homology. There are a number of algorithms, and computerprograms based thereon, which are available to be used the literatureand/or publically or commercially available for performing alignmentsand percent identity. The selection of the algorithm or program is not alimitation of the present invention.

Examples of suitable alignment programs including, e.g., the softwareCLUSTALW under Unix and then be imported into the Bioedit program (Hall,T. A. 1999, BioEdit: a user-friendly biological sequence alignmenteditor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp.Ser. 41:95-98); the Clustal Omega available from EMBL-EBI (Sievers,Fabian, et al. “Fast, scalable generation of high-quality proteinmultiple sequence alignments using Clustal Omega.” Molecular systemsbiology 7.1 (2011): 539 and Goujon, Mickael, et al. “A newbioinformatics analysis tools framework at EMBL-EBI.” Nucleic acidsresearch 38.suppl 2 (2010): W695-W699); the Wisconsin Sequence AnalysisPackage, version 9.1 (Devereux J. et al., Nucleic Acids Res.,12:387-395, 1984, available from Genetics Computer Group, Madison, Wis.,USA). The programs BESTFIT and GAP, may be used to determine the %identity between two polynucleotides and the % identity between twopolypeptide sequences.

Other programs for determining identity and/or similarity betweensequences include, e.g, the BLAST family of programs available from theNational Center for Biotechnology Information (NCB), Bethesda, Md., USAand accessible through the home page of the NCBI atwww.ncbi.nlm.nih.gov), the ALIGN program (version 2.0) which is part ofthe GCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used;and FASTA (Pearson W. R. and Lipman D. J., Proc. Natl. Acad. Sci. USA,85:2444-2448, 1988, available as part of the Wisconsin Sequence AnalysisPackage). SeqWeb Software (a web-based interface to the GCG WisconsinPackage: Gap program).

In some embodiments, the cassette is designed to be expressed from arecombinant adeno-associated virus, and the vector genome also containsAAV inverted terminal repeats (ITRs). In one embodiment, the rAAV ispseudotyped, i.e., the AAV capsid is from a different source AAV thanthat the AAV which provides the ITRs. In one embodiment, the ITRs of AAVserotype 2 are used. However, ITRs from other suitable sources may beselected. Optionally, the AAV may be a self-complementary AAV.

The expression cassettes described herein utilized AAV 5′ invertedterminal repeat (ITR) and an AAV 3′ ITR. However, other configurationsof these elements may be suitable. A shortened version of the 5′ ITR,termed ΔITR, has been described in which the D-sequence and terminalresolution site (trs) are deleted. In other embodiments, the full-lengthAAV 5′ and/or 3′ ITRs are used. Where a pseudotyped AAV is to beproduced, the ITRs in the expression are selected from a source whichdiffers from the AAV source of the capsid. For example, AAV2 ITRs may beselected for use with an AAV capsid having a particular efficiency fortargeting CNS or tissues or cells within the CNS. In one embodiment, theITR sequences from AAV2, or the deleted version thereof (ΔITR), are usedfor convenience and to accelerate regulatory approval. However, ITRsfrom other AAV sources may be selected. Where the source of the ITRs isfrom AAV2 and the AAV capsid is from another AAV source, the resultingvector may be termed pseudotyped. However, other sources of AAV ITRs maybe utilized.

In one embodiment, the expression cassette is designed for expressionand secretion in the central nervous system (CNS), including thecerebral spinal fluid and brain. In a particularly desired embodiment,the expression cassette is useful for expression in both the CNS and inthe liver, thereby allowing treatment of both the systemic andCNS-related effects of MPS II. For example, the inventors have observedthat certain constitutive promoters (e.g., CMV) do not drive expressionat desired levels when delivered intrathecally, thereby providingsuboptimal hIDS expression levels. However, the chicken beta-actinpromoter drives expression well both upon intrathecal delivery andsystemic delivery. Thus, this is a particularly desirable promoter.Other promoters may be selected, but expression cassettes containingsame may not have all of the advantages of those with a chickenbeta-actin promoter. A variety of chicken beta-actin promoters have beendescribed alone, or in combination with various enhancer elements (e.g.,CB7 is a chicken beta-actin promoter with cytomegalovirus enhancerelements, a CAG promoter, which includes the promoter, the first exonand first intron of chicken beta actin, and the splice acceptor of therabbit beta-globin gene), a CBh promoter [S J Gray et al, Hu Gene Ther,2011 September; 22(9): 1143-1153.

Examples of promoters that are tissue-specific are well known for liverand other tissues (albumin, Miyatake et al., (1997) J. Virol.,71:5124-32; hepatitis B virus core promoter, Sandig et al., (1996) GeneTher., 3:1002-9; alpha-fetoprotein (AFP), Arbuthnot et al., (1996) Hum.Gene Ther., 7:1503-14), bone osteocalcin (Stein et al., (1997) Mol.Biol. REP., 24:185-96); bone sialoprotein (Chen et al., (1996) J. BoneMiner. Res., 11:654-64), lymphocytes (CD2, Hansal et al., (1998) J.Immunol., 161:1063-8; immunoglobulin heavy chain; T cell receptorchain), neuronal such as neuron-specific enolase (NSE) promoter(Andersen et al., (1993) Cell. Mol. Neurobiol., 13:503-15),neurofilament light-chain gene (Piccioli et al., (1991) Proc. Natl.Acad. Sci. USA, 88:5611-5), and the neuron-specific vgf gene (Piccioliet al., (1995) Neuron, 15:373-84), among others. Alternatively, aregulatable promoter may be selected. See, e.g., WO 2011/126808B2,incorporated by reference herein.

In one embodiment, the expression cassette comprises one or moreexpression enhancers. In one embodiment, the expression cassettecontains two or more expression enhancers. These enhancers may be thesame or may be different. For example, an enhancer may include an Alphamic/bik enhancer or a CMV enhancer. This enhancer may be present in twocopies which are located adjacent to one another. Alternatively, thedual copies of the enhancer may be separated by one or more sequences.In still another embodiment, the expression cassette further contains anintron, e.g, a chicken beta-actin intron, a human β-globulin intron,and/or a commercially available Promega® intron. Other suitable intronsinclude those known in the art, e.g., such as are described in WO2011/126808.

Further, an expression cassette is provided with a suitablepolyadenylation signal. In one embodiment, the polyA sequence is arabbit globulin poly A. See, e.g., WO 2014/151341. Alternatively,another polyA, e.g., a human growth hormone (hGH) polyadenylationsequence, an SV40 poly A, SV50 polyA, or a synthetic polyA. Still otherconventional regulatory elements may be additional or optionallyincluded in an expression cassette.

An exemplary rAAV.hIDS vector genome is shown in nt 2 to nt 3967 of SEQID NO: 3, nt 11 to nt 4964 of SEQ ID NO: 5, nt 11 to nt 4964 of SEQ IDNO: 8, nt 2 to nt 3967 of SEQ ID NO: 11, or nt 2 to nt 3965 of SEQ IDNO: 14.

5.1.2. Production of rAAV.hIDS Viral Particles

In one embodiment, a recombinant adeno-associated virus (rAAV) particlehaving an AAV capsid and having packaged therein a AAV inverted terminalrepeats, a human iduronate-2-sulfatase (hIDS) gene under the control ofregulatory sequences which control expression thereof, wherein said hIDSgene has a sequence shown in SEQ ID NO: 1 (FIG. 1) or a sequence atleast about 95% identical thereto which encodes a functional humaniduronate-2-sulfatase is provided. In one embodiment, the hIDSexpression cassette is flanked by an AAVS' ITR and an AAV3′ ITR. Inanother embodiment, the AAV is a single stranded AAV.

For intrathecal and/or intracisternal delivery, AAV9 is particularlydesirable. Optionally, an rAAV9.hIDS vector as described herein may beco-administered with a vector designed to specifically target the liver.Any of a number of rAAV vectors with liver tropism can be used. Examplesof AAV which may be selected as sources for capsids of rAAV include,e.g., rh10, AAVrh64R1, AAVrh64R2, rh8 [See, e.g., US Published PatentApplication No. 2007-0036760-A1; US Published Patent Application No.2009-0197338-A1; EP 1310571]. See also, WO 2003/042397 (AAV7 and othersimian AAV), U.S. Pat. Nos. 7,790,449 and 7,282,199 (AAV8), WO2005/033321 and U.S. Pat. No. 7,906,111 (AAV9), and WO 2006/110689], andrh10 [WO 2003/042397], AAV3B; AAVdj [US 2010/0047174]. One particularlydesirable rAAV is AAV2/8.TBG.hIDS.co.

In many instances, rAAV particles are referred to as DNase resistant.However, in addition to this endonuclease (DNase), other endo- andexo-nucleases may also be used in the purification steps describedherein, to remove contaminating nucleic acids. Such nucleases may beselected to degrade single stranded DNA and/or double-stranded DNA, andRNA. Such steps may contain a single nuclease, or mixtures of nucleasesdirected to different targets, and may be endonucleases or exonucleases.

Methods of preparing AAV-based vectors are known. See, e.g., USPublished Patent Application No. 2007/0036760 (Feb. 15, 2007), which isincorporated by reference herein. The use of AAV capsids of AAV9 areparticularly well suited for the compositions and methods describedherein. The sequences of AAV9 and methods of generating vectors based onthe AAV9 capsid are described in U.S. Pat. No. 7,906,111;US2015/0315612; WO 2012/112832; which are incorporated herein byreference. However, other AAV capsids may be selected or generated. Forexample, the sequences of AAV8 and methods of generating vectors basedon the AAV8 capsid are described in U.S. Pat. No. 7,282,199 B2, U.S.Pat. Nos. 7,790,449, and 8,318,480, which are incorporated herein byreference. The sequences of a number of such AAV are provided in theabove-cited U.S. Pat. No. 7,282,199 B2, U.S. Pat. Nos. 7,790,449,8,318,480, and 7,906,111, and/or are available from GenBank. Thesequences of any of the AAV capsids can be readily generatedsynthetically or using a variety of molecular biology and geneticengineering techniques. Suitable production techniques are well known tothose of skill in the art. See, e.g., Sambrook et al, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor,N.Y.). Alternatively, oligonucleotides encoding peptides (e.g., CDRs) orthe peptides themselves can generated synthetically, e.g., by thewell-known solid phase peptide synthesis methods (Merrifield, (1962) J.Am. Chem. Soc., 85:2149; Stewart and Young, Solid Phase PeptideSynthesis (Freeman, San Francisco, 1969) pp. 27-62). These and othersuitable production methods are within the knowledge of those of skillin the art and are not a limitation of the present invention.

The recombinant adeno-associated virus (AAV) described herein may begenerated using techniques which are known. See, e.g., WO 2003/042397;WO 2005/033321, WO 2006/110689; U.S. Pat. No. 7,588,772 B2. Such amethod involves culturing a host cell which contains a nucleic acidsequence encoding an AAV capsid; a functional rep gene; an expressioncassette composed of, at a minimum, AAV inverted terminal repeats (ITRs)and a transgene; and sufficient helper functions to permit packaging ofthe expression cassette into the AAV capsid protein.

To calculate empty and full particle content, VP3 band volumes for aselected sample (e.g., in examples herein an iodixanol gradient-purifiedpreparation where # of GC=# of particles) are plotted against GCparticles loaded. The resulting linear equation (y=mx+c) is used tocalculate the number of particles in the band volumes of the testarticle peaks. The number of particles (pt) per 20 μL loaded is thenmultiplied by 50 to give particles (pt)/mL. Pt/mL divided by GC/mL givesthe ratio of particles to genome copies (pt/GC). Pt/mL-GC/mL gives emptypt/mL. Empty pt/mL divided by pt/mL and ×100 gives the percentage ofempty particles.

Generally, methods for assaying for empty capsids and AAV vectorparticles with packaged genomes have been known in the art. See, e.g.,Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et al., Molec.Ther. (2003) 7:122-128. To test for denatured capsid, the methodsinclude subjecting the treated AAV stock to SDS-polyacrylamide gelelectrophoresis, consisting of any gel capable of separating the threecapsid proteins, for example, a gradient gel containing 3-8%Tris-acetate in the buffer, then running the gel until sample materialis separated, and blotting the gel onto nylon or nitrocellulosemembranes, preferably nylon. Anti-AAV capsid antibodies are then used asthe primary antibodies that bind to denatured capsid proteins,preferably an anti-AAV capsid monoclonal antibody, most preferably theB1 anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000)74:9281-9293). A secondary antibody is then used, one that binds to theprimary antibody and contains a means for detecting binding with theprimary antibody, more preferably an anti-IgG antibody containing adetection molecule covalently bound to it, most preferably a sheepanti-mouse IgG antibody covalently linked to horseradish peroxidase. Amethod for detecting binding is used to semi-quantitatively determinebinding between the primary and secondary antibodies, preferably adetection method capable of detecting radioactive isotope emissions,electromagnetic radiation, or colorimetric changes, most preferably achemiluminescence detection kit. For example, for SDS-PAGE, samples fromcolumn fractions can be taken and heated in SDS-PAGE loading buffercontaining reducing agent (e.g., DTT), and capsid proteins were resolvedon pre-cast gradient polyacrylamide gels (e.g., Novex). Silver stainingmay be performed using SilverXpress (Invitrogen, CA) according to themanufacturer's instructions or other suitable staining method, i.e.SYPRO ruby or coomassie stains. In one embodiment, the concentration ofAAV vector genomes (vg) in column fractions can be measured byquantitative real time PCR (Q-PCR). Samples are diluted and digestedwith DNase I (or another suitable nuclease) to remove exogenous DNA.After inactivation of the nuclease, the samples are further diluted andamplified using primers and a TaqMan™ fluorogenic probe specific for theDNA sequence between the primers. The number of cycles required to reacha defined level of fluorescence (threshold cycle, Ct) is measured foreach sample on an Applied Biosystems Prism 7700 Sequence DetectionSystem. Plasmid DNA containing identical sequences to that contained inthe AAV vector is employed to generate a standard curve in the Q-PCRreaction. The cycle threshold (Ct) values obtained from the samples areused to determine vector genome titer by normalizing it to the Ct valueof the plasmid standard curve. End-point assays based on the digital PCRcan also be used.

In one aspect, an optimized q-PCR method is used which utilizes a broadspectrum serine protease, e.g., proteinase K (such as is commerciallyavailable from Qiagen). More particularly, the optimized qPCR genometiter assay is similar to a standard assay, except that after the DNaseI digestion, samples are diluted with proteinase K buffer and treatedwith proteinase K followed by heat inactivation. Suitably samples arediluted with proteinase K buffer in an amount equal to the sample size.The proteinase K buffer may be concentrated to 2 fold or higher.Typically, proteinase K treatment is about 0.2 mg/mL, but may be variedfrom 0.1 mg/mL to about 1 mg/mL. The treatment step is generallyconducted at about 55° C. for about 15 minutes, but may be performed ata lower temperature (e.g., about 37° C. to about 50° C.) over a longertime period (e.g., about 20 minutes to about 30 minutes), or a highertemperature (e.g., up to about 60° C.) for a shorter time period (e.g.,about 5 to 10 minutes). Similarly, heat inactivation is generally atabout 95° C. for about 15 minutes, but the temperature may be lowered(e.g., about 70 to about 90° C.) and the time extended (e.g., about 20minutes to about 30 minutes). Samples are then diluted (e.g., 1000 fold)and subjected to TaqMan analysis as described in the standard assay.

Additionally, or alternatively, droplet digital PCR (ddPCR) may be used.For example, methods for determining single-stranded andself-complementary AAV vector genome titers by ddPCR have beendescribed. See, e.g., M. Lock et al, Hu Gene Therapy Methods, Hum GeneTher Methods. 2014 April; 25(2):115-25. doi: 10.1089/hgtb.2013.131. Epub2014 Feb. 14.

In brief, the method for separating rAAV9 particles having packagedgenomic sequences from genome-deficient AAV9 intermediates involvessubjecting a suspension comprising recombinant AAV9 viral particles andAAV 9 capsid intermediates to fast performance liquid chromatography,wherein the AAV9 viral particles and AAV9 intermediates are bound to astrong anion exchange resin equilibrated at a pH of 10.2, and subjectedto a salt gradient while monitoring eluate for ultraviolet absorbance atabout 260 and about 280. Although less optimal for rAAV9, the pH may bein the range of about 10.0 to 10.4. In this method, the AAV9 fullcapsids are collected from a fraction which is eluted when the ratio ofA260/A280 reaches an inflection point. In one example, for the AffinityChromatography step, the diafiltered product may be applied to a CaptureSelect™ Poros-AAV2/9 affinity resin (Life Technologies) that efficientlycaptures the AAV2/9 serotype. Under these ionic conditions, asignificant percentage of residual cellular DNA and proteins flowthrough the column, while AAV particles are efficiently captured.

The rAAV.hIDS vector can be manufactured as shown in the flow diagramshown in FIG. 11, which is described in more detail in Section 5.4 andExample 4, infra.

5.1.3. Pharmaceutical Formulations of rAAV.hIDS

The rAAV9.hIDS formulation is a suspension containing an effectiveamount of AAV.hIDS vector suspended in an aqueous solution containingsaline, a surfactant, and a physiologically compatible salt or mixtureof salts. Suitably, the formulation is adjusted to a physiologicallyacceptable pH, e.g., in the range of pH 6 to 9, or pH 6.5 to 7.5, pH 7.0to 7.7, or pH 7.2 to 7.8. As the pH of the cerebrospinal fluid is about7.28 to about 7.32, for intrathecal delivery, a pH within this range maybe desired; whereas for intravenous delivery, a pH of 6.8 to about 7.2may be desired. However, other pHs within the broadest ranges and thesesubranges may be selected for other route of delivery.

A suitable surfactant, or combination of surfactants, may be selectedfrom among non-ionic surfactants that are nontoxic. In one embodiment, adifunctional block copolymer surfactant terminating in primary hydroxylgroups is selected, e.g., such as Pluronic® F68 [BASF], also known asPoloxamer 188, which has a neutral pH, has an average molecular weightof 8400. Other surfactants and other Poloxamers may be selected, i.e.,nonionic triblock copolymers composed of a central hydrophobic chain ofpolyoxypropylene (poly(propylene oxide)) flanked by two hydrophilicchains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15(Macrogol-15 Hydroxystearate), LABRASOL (Polyoxy capryllic glyceride),polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acidesters), ethanol and polyethylene glycol. In one embodiment, theformulation contains a poloxamer. These copolymers are commonly namedwith the letter “P” (for poloxamer) followed by three digits: the firsttwo digits ×100 give the approximate molecular mass of thepolyoxypropylene core, and the last digit ×10 gives the percentagepolyoxyethylene content. In one embodiment Poloxamer 188 is selected.The surfactant may be present in an amount up to about 0.0005% to about0.001% of the suspension.

In one embodiment, the formulation may contain, e.g., a concentration ofat least about 1×10⁹ GC/mL to 3×10¹³ GC/mL as measured by qPCR ordigital droplet PCR (ddPCR) as described in, e.g., M. Lock et al, HuGene Therapy Methods, Hum Gene Ther Methods. 2014 April; 25(2):115-25.doi: 10.1089/hgtb.2013.131. Epub 2014 Feb. 14, which is incorporatedherein by reference.

In one embodiment, a frozen composition which contains an rAAV in abuffer solution as described herein, in frozen form, is provided.Optionally, one or more surfactants (e.g., Pluronic F68), stabilizers orpreservatives is present in this composition. Suitably, for use, acomposition is thawed and titrated to the desired dose with a suitablediluent, e.g., sterile saline or a buffered saline.

In one example, the formulation may contain, e.g., buffered salinesolution comprising one or more of sodium chloride, sodium bicarbonate,dextrose, magnesium sulfate (e.g., magnesium sulfate.7H₂O), potassiumchloride, calcium chloride (e.g., calcium chloride.2H₂O), dibasic sodiumphosphate, and mixtures thereof, in water. Suitably, for intrathecaldelivery, the osmolarity is within a range compatible with cerebrospinalfluid (e.g., about 275 to about 290); see, e.g.,http://emedicine.medscape.com/article/2093316-overview. Optionally, forintrathecal delivery, a commercially available diluent may be used as asuspending agent, or in combination with another suspending agent andother optional excipients. See, e.g., Elliotts B® solution [LukareMedical]. In other embodiments, the formulation may contain one or morepermeation enhancers. Examples of suitable permeation enhancers mayinclude, e.g., mannitol, sodium glycocholate, sodium taurocholate,sodium deoxycholate, sodium salicylate, sodium caprylate, sodiumcaprate, sodium lauryl sulfate, polyoxyethylene-9-laurel ether, or EDTA.

In certain embodiments, a kit is provided which includes a concentratedvector suspended in a formulation (optionally frozen), optional dilutionbuffer, and devices and other components required for intrathecaladministration are provided. In another embodiment, the kit mayadditional or alternatively include components for intravenous delivery.In one embodiment, the kit provides sufficient buffer to allow forinjection. Such buffer may allow for about a 1:1 to a 1:5 dilution ofthe concentrated vector, or more. In other embodiments, higher or loweramounts of buffer or sterile water are included to allow for dosetitration and other adjustments by the treating clinician. In stillother embodiments, one or more components of the device are included inthe kit.

5.2. Gene Therapy Protocol

5.2.1 Target Patient Populations

Provided herein are methods for treating type II mucopolysaccharidosiscomprising delivering a therapeutically effective amount of a rAAV.hIDSdescribed herein to a patient in need thereof. In particular, providedherein are methods for preventing, treating, and/or amelioratingneurocognitive decline in a patient diagnosed with MPS II, comprisingdelivering a therapeutically effective amount of a rAAV.hIDS describedherein to a patient in need thereof. A “therapeutically effectiveamount” of the rAAV.hIDS vector described herein may correct one or moreof the symptoms identified in any one of the following paragraphs.

Patients who are candidates for treatment are pediatric and adultpatients with MPS II (Hunter syndrome) and/or the symptoms associatedwith MPS II. MPS II is characterized as a mild/attenuated phenotype or asevere phenotype. Death occurs at a mean age of 11.7 years in patientswith the severe phenotype (characterized by neurocognitivedeterioration) and 21.7 years in patients with a mild or attenuatedphenotype. The majority (two-thirds) of patients have the severe form ofthis disease. Patients with MPS II appear normal at birth, but signs andsymptoms of disease typically present between the ages of 18 months and4 years in the severe form and between the ages of 4 and 8 years in theattenuated form. Signs and symptoms common to all affected patientsinclude short stature, coarse facial features, macrocephaly,macroglossia, hearing loss, hepato- and splenomegaly, dystosismultiplex, joint contractures, spinal stenosis and carpal tunnelsyndrome. Frequent upper respiratory and ear infections occur in mostpatients and progressive airway obstruction is commonly found, leadingto sleep apnea and often death. Cardiac disease is a major cause ofdeath in this population and is characterized by valvular dysfunctionleading to right and left ventricular hypertrophy and heart failure.Death is generally attributed to obstructive airway disease or cardiacfailure.

In severe forms of MPS II, early developmental milestones may be met,but developmental delay is readily apparent by 18-24 months. Somepatients fail hearing screening tests in the first year and othermilestones are delayed, including ability to sit unsupported, ability towalk, and speech. Developmental progression begins to plateau between 3and 5 years of age, with regression reported to begin around 6.5 years.Of the ˜50% of children with MPS II who become toilet trained, most, ifnot all, will lose this ability as the disease progresses.

Patients with significant neurologic involvement exhibit severebehavioral disturbances including hyperactivity, obstinacy, andaggression beginning in the second year of life and continuing until age8-9, when neurodegeneration attenuates this behavior.

Seizures are reported in over half of severely affected patients whoreach the age of 10, and by the time of death most patients with CNSinvolvement are severely mentally handicapped and require constant care.Although patients with attenuated disease exhibit normal intellectualfunctioning, MRI imaging reveals gross brain abnormalities in allpatients with MPS II including white matter lesions, enlargedventricles, and brain atrophy.

A composition of the present invention avoids complications of long-termenzyme replacement therapy (ERT) related to immune response to therecombinant enzyme which can range from mild to full-blown anaphylaxisas well as complications of life-long peripheral access such as localand systemic infections. In contrast to ERT, a composition of theinvention does not require life-long, repeated weekly injections.Without wishing to be bound by theory, the therapeutic method describedherein is believed to be useful for correcting at least the centralnervous system phenotype associated with MPS II disorders by providingefficient, long-term gene transfer afforded by vectors with hightransduction efficiency which provide continuous, elevated circulatingIDS levels, which provides therapeutic leverage outside the CNScompartment. In addition, provided herein are methods for providingactive tolerance and preventing antibody formation against the enzyme bya variety of routes, including by direct systemic delivery of the enzymein protein form or in the form of AAV-hIDS prior to AAV-mediateddelivery into CNS.

In some embodiments, patients diagnosed with severe MPS II are treatedin accordance with the methods described herein. In some embodiments,patients diagnosed with attenuated MPS II are treated in accordance withthe methods described herein. In some embodiments, pediatric subjectswith MPS II who have an early-stage neurocognitive deficit are treatedin accordance with the methods described herein. In certain embodiments,patients age 2 years or older diagnosed with Hunter syndrome and havingneurocognitive deficits or at risk of developing neurocognitive deficitsare treated in accordance with the methods described herein. In certainembodiments, patients age 2 years or older with the presence of a majorrearrangement or deletion mutation that is known to correlate withsevere Hunter syndrome are treated in accordance with the methodsdescribed herein.

In certain embodiments, newborn babies (3 months old or younger) aretreated in accordance with the methods described herein. In certainembodiments, babies that are 3 months old to 9 months old are treated inaccordance with the methods described herein. In certain embodiments,children that are 9 months old to 36 months old are treated inaccordance with the methods described herein. In certain embodiments,children that are 3 years old to 12 years old are treated in accordancewith the methods described herein. In certain embodiments, children thatare 12 years old to 18 years old are treated in accordance with themethods described herein. In certain embodiments, adults that are 18years old or older are treated in accordance with the methods describedherein.

Suitably, patients selected for treatment may include those having oneor more of the following characteristics: a documented diagnosis of MPSII confirmed by the lacking or diminished IDS enzyme activity asmeasured in serum, plasma, fibroblasts, or leukocytes; documentedevidence of early-stage neurocognitive deficit due to MPS II, defined aseither of the following, if not explainable by any other neurological orpsychiatric factors:—a DQ (BSID-III) that is at least 1 standarddeviation below mean or documented historical evidence of a decline ofgreater than 1 standard deviation on sequential testing. Alternatively,increased GAGs in urine, serum, CSF, or genetic tests may be used.

Prior to treatment, subjects, e.g., infants, preferably undergogenotyping to identify MPS II patients, i.e., patients that havemutations in the gene encoding hIDS. Prior to treatment, the MPS IIpatient can be assessed for neutralizing antibodies (Nab) to the AAVserotype used to deliver the hIDS gene. Such Nabs can interfere withtransduction efficiency and reduce therapeutic efficacy. MPS II patientsthat have a baseline serum Nab titer ≤1:5 are good candidates fortreatment with the rAAV.hIDS gene therapy protocol. Treatment of MPS IIpatients with titers of serum Nab>1:5 may require a combination therapy,such as transient co-treatment with an immunosuppressant before and/orduring treatment with rAAV.hIDS vector delivery. Optionally,immunosuppressive co-therapy may be used as a precautionary measurewithout prior assessment of neutralizing antibodies to the AAV vectorcapsid and/or other components of the formulation. Priorimmunosuppression therapy may be desirable to prevent potential adverseimmune reaction to the hIDS transgene product, especially in patientswho have virtually no levels of IDUA activity, where the transgeneproduct may be seen as “foreign.” Results of non-clinical studies inmice, dogs and NHPs described infra are consistent with the developmentof an immune response to hIDS and neuroinflammation. While a similarreaction may not occur in human subjects, as a precautionimmunosuppression therapy is recommended for all recipients ofrAAV-hIDS.

Immunosuppressants for such co-therapy include, but are not limited to,a glucocorticoid, steroids, antimetabolites, T-cell inhibitors, amacrolide (e.g., a rapamycin or rapalog), and cytostatic agentsincluding an alkylating agent, an anti-metabolite, a cytotoxicantibiotic, an antibody, or an agent active on immunophilin. The immunesuppressant may include a nitrogen mustard, nitrosourea, platinumcompound, methotrexate, azathioprine, mercaptopurine, fluorouracil,dactinomycin, an anthracycline, mitomycin C, bleomycin, mithramycin,IL-2 receptor- (CD25-) or CD3-directed antibodies, anti-IL-2 antibodies,cyclosporin, tacrolimus, sirolimus, IFN-β, IFN-γ, an opioid, or TNF-α(tumor necrosis factor-alpha) binding agent. In certain embodiments, theimmunosuppressive therapy may be started 0, 1, 2, 7, or more days priorto the gene therapy administration. Such therapy may involveco-administration of two or more drugs, the (e.g., prednelisone,micophenolate mofetil (MMF) and/or sirolimus (i.e., rapamycin)) on thesame day. One or more of these drugs may be continued after gene therapyadministration, at the same dose or an adjusted dose. Such therapy maybe for about 1 week (7 days), about 60 days, or longer, as needed. Incertain embodiments, a tacrolimus-free regimen is selected.

In certain embodiments, patients having one or more of the followingcharacteristics may be excluded from treatment at the discretion oftheir caring physician:

-   -   Has any neurocognitive deficit not attributable to MPS II that        may in the opinion of either the investigator or the medical        monitor confound interpretation of study results    -   Has any condition (e.g., history of any disease, evidence of any        current disease, any finding upon physical examination, or any        laboratory abnormality) that, in the opinion of the        investigator, would put the subject at undue risk or would        interfere with evaluation of the investigational product or        interpretation of subject safety or study results    -   Diagnosis of neuropsychiatric condition    -   Has any contraindication to intrathecal/intracranial treatment        administration, including contraindications to fluoroscopic        imaging    -   Has any contraindication to MRI    -   Has acute hydrocephalus at time of enrollment    -   Is currently enrolled in any other clinical study with an        investigational product within 4 weeks prior to Screening or        within 5 half-lives of the investigational product used in that        clinical study, whichever is longer    -   Has undergone hematopoietic stem cell transplantation (HSCT)    -   Has received idursulfase via intrathecal administration within 6        months prior to screening    -   Has received intrathecal idursulfase at any time and experienced        a significant adverse effect (AE) considered related to        intrathecal administration that in opinion of the investigator        and/or medical monitor would put the subject at undue risk.

In other embodiments, a caring physician may determine that the presenceof one or more of these physical characteristics (medical history)should not preclude treatment as provided herein.

5.2.2. Dosages & Mode of Administration

Pharmaceutical compositions suitable for administration to patientscomprise a suspension of rAAV.hIDS vectors in a formulation buffercomprising a physiologically compatible aqueous buffer, a surfactant andoptional excipients. In certain embodiments, a pharmaceuticalcomposition described herein is administered intrathecally. In otherembodiments, a pharmaceutical composition described herein isadministered intracisternally. In other embodiments, a pharmaceuticalcomposition described herein is administered intravenously. In certainembodiments, the pharmaceutical composition is delivered via aperipheral vein by infusion over 20 minutes (±5 minutes). However, thistime may be adjusted as needed or desired. However, still other routesof administration may be selected. Alternatively or additionally, routesof administration may be combined, if desired.

While a single administration of the rAAV is anticipated to beeffective, administration may be repeated (e.g., quarterly, bi-annually,annually, or as otherwise needed, particularly in treatment of newborns.Optionally, an initial dose of a therapeutically effective amount may bedelivered over split infusion/injection sessions, taking intoconsideration the age and ability of the subject to tolerateinfusions/injections. However, repeated weekly injections of a fulltherapeutic dose are not required, providing an advantage to the patientin terms of both comfort and therapeutic outcome.

In some embodiments, the rAAV suspension has an rAAV Genome Copy (GC)titer that is at least 1.0×10¹³ GC/mL. In certain embodiments, the rAAVEmpty/Full particle ratio in the rAAV suspension is between 0.01 and0.05 (95%-99% free of empty capsids). In some embodiments, an MPS IIpatient in need thereof is administered a dose of at least about 4×10⁸GC/g brain mass to about 4×10¹¹ GC/g brain mass of the rAAV suspension.

The following therapeutically effective flat doses of rAAV.hIDS can beadministered to MPS II patients of the indicated age group:

Newborns: about 3.8×10¹² to about 1.9×10¹⁴ GC;

3-9 months: about 6×10¹² to about 3×10¹⁴ GC;

9-36 months: about 10¹³ to about 5×10¹⁴ GC;

3-12 years: about 1.2×10¹³ to about 6×10¹⁴ GC;

12+ years: about 1.4×10¹³ to about 7.0×10¹⁴ GC;

18+ years (adult): about 1.4×10¹³ to about 7.0×10¹⁴ GC.

In some embodiments, the dose administered to a 12+ year old MPS IIpatient (including 18+ year old) is 1.4×10¹³ genome copies (GC)(1.1×10¹⁰ GC/g brain mass). In some embodiments, the dose administeredto a 12+ year old MPS II patient (including 18+ year old) is 7×10¹³ GC(5.6×10¹⁰ GC/g brain mass). In still a further embodiment, the doseadministered to an MPS II patient is at least about 4×10⁸ GC/g brainmass to about 4×10¹¹ GC/g brain mass. In certain embodiments, the doseadministered to MPS II newborns ranges from about 1.4×10¹¹ to about1.4×10¹⁴ GC; the dose administered to infants 3-9 months ranges fromabout 2.4×10¹¹ to about 2.4×10¹⁴ GC; the dose administered to MPS IIchildren 9-36 months ranges: about 4×10¹¹ to about 4×10¹⁴ GC; the doseadministered to MPS II children 3-12 years: ranges from about 4.8×10¹¹to about 4.8×10¹⁴ GC; the dose administered to children and adults 12+years ranges from about 5.6×10¹¹ to about 5.6×10¹⁴ GC.

Suitable volumes for delivery of these doses and concentrations may bedetermined by one of skill in the art. For example, volumes of about 1μL to 150 mL may be selected, with the higher volumes being selected foradults. Typically, for newborn infants a suitable volume is about 0.5 mLto about 10 mL, for older infants, about 0.5 mL to about 15 mL may beselected. For toddlers, a volume of about 0.5 mL to about 20 mL may beselected. For children, volumes of up to about 30 mL may be selected.For pre-teens and teens, volumes up to about 50 mL may be selected. Instill other embodiments, a patient may receive an intrathecaladministration in a volume of about 5 mL to about 15 mL are selected, orabout 7.5 mL to about 10 mL. Other suitable volumes and dosages may bedetermined. The dosage will be adjusted to balance the therapeuticbenefit against any side effects and such dosages may vary dependingupon the therapeutic application for which the recombinant vector isemployed.

5.2.3. Monitoring Efficacy

Efficacy of the therapy described herein can be measured by assessing(a) the prevention of neurocognitive decline in patients with MPS II(Hunter syndrome); and (b) reductions in biomarkers of disease, e.g.,GAG levels and/or enzyme activity in the CSF, serum and/or urine, and/orliver and spleen volumes. Neurocognition can be determined by measuringintelligence quotient (IQ), e.g., as measured by Bayley's InfantileDevelopment Scale for Hurler subjects or as measured by the WechslerAbbreviated Scale of Intelligence (WASI) for Hurler-Scheie subjects.Other appropriate measures of neurocognitive development and functionmay be utilized, e.g., assessing developmental quotient (DQ) usingBayley Scales of Infant Development (BSID-III), assessing memory usingthe Hopkins Verbal Learning Test, and/or using Tests of Variables ofAttention (TOVA). Other neuropsychological function, such as vinelandadaptive behavior scales, visual processing, fine motor, communication,socialization, daily living skills, and emotional and behavioral healthare monitored. Magnetic Resonance Imaging (MRI) of brain to acquirevolumetric, diffusion tensor imaging (DTI), and resting state data,median nerve cross-sectional area by ultrasonography, improvement inspinal cord compression, safety, liver size and spleen size are alsoadministered.

Optionally, other measures of efficacy may include evaluation ofbiomarkers (e.g., polyamines as described herein) and clinical outcomes.Urine is evaluated for total GAG content, concentration of GAG relativeto creatinine, as well as MPS II specific pGAGs. Serum and/or plasma isevaluated for IDS activity, anti-IDS antibodies, pGAG, and concentrationof the heparin cofactor II-thrombin complex and markers of inflammation.CSF is evaluated for IDUA activity, anti-IDS antibodies, hexosaminidase(hex) activity, and pGAG (such as heparan sulfate and dermatan sulfate).The presence of neutralizing antibodies to vector (e.g., AAV9) andbinding antibodies to anti-IDS antibodies may be assessed in CSF andserum. T-cell response to vector capsid (e.g., AAV9) or the hIDStransgene product may be assessed by ELISPOT assay. Pharmacokinetics ofIDS expression in CSF, serum, and urine as well as vector concentration(PCR to AAV9 DNA) may also be monitored.

Combinations of gene therapy delivery of the rAAV.hIDS to the CNSaccompanied by systemic delivery of hIDS are encompassed by the methodsof the invention. Systemic delivery can be accomplished using ERT (e.g.,using Elaprase® (idursulfase)), or additional gene therapy using anrAAV.hIDS with tropism for the liver (e.g., an rAAV.hIDS bearing an AAV8capsid).

Additional measures of clinical efficacy associated with systemicdelivery may include, e.g., Orthopedic Measures, such as bone mineraldensity, bone mineral content, bone geometry and strength, Bone Densitymeasured by dual energy x-ray absorptiometry (DXA); Height (Z-scores forstanding height/lying-length-for-age); Markers of Bone Metabolism:Measurements of Serum osteocalcin (OCN) and bone-specific alkalinephosphatase (BSAP), carboxy terminal telopeptide of type I collagen(ICTP) and carboxy terminal telopeptide al chain of type I collagen(CTX); Flexibility and Muscle Strength: Biodex and Physical Therapyevaluations, including 6 minute walk study (The Biodex III isokineticstrength testing system is used to assess strength at the knee and elbowfor each participant); Active Joint Range of Motion (ROM); Child HealthAssessment Questionnaire/Health Assessment Questionnaire (CHAQ/HAQ)Disability Index Score; Electromyographic (EMG) and/or OxygenUtilization to Monitor an individual's cardiorespiratory fitness: peakoxygen uptake (VO₂ peak) during exercise testing; Apnea/Hypopnea Index(AHI); Forced Vital Capacity (FVC); Left Ventricular Mass (LVM).

In certain embodiments, a method of diagnosing and/or treating MPS II ina patient, or monitoring treatment, is provided. The method involvesobtaining a cerebrospinal fluid or plasma sample from a human patientsuspected of having MPS II; detecting spermine concentration levels inthe sample; diagnosing the patient with a mucopolysaccharidosis selectedfrom MPS II in the patient having spermine concentrations in excess of 1ng/mL; and delivering an effective amount of human IDS to the diagnosedpatient as provided herein.

In another aspect, the method involves monitoring and adjusting MPS IItherapy. Such method involves obtaining a cerebrospinal fluid or plasmasample from a human patient undergoing therapy for MPS II; detectingspermine concentration levels in the sample by performing a massspectral analysis; adjusting dosing levels of the MPS II therapeutic.For example, “normal” human spermine concentrations are about 1 ng/mL to2 ng/mL or less in cerebrospinal fluid. However, patients havinguntreated MPS II may have spermine concentration levels of greater than2 ng/mL and up to about 100 ng/mL. If a patient has levels approachingnormal levels, dosing of any companion may be lowered. Conversely, if apatient has higher than desired MPS II levels, higher doses, or anadditional therapy, e.g., ERT, may be provided to the patient.

Spermine concentration may be determined using a suitable assay. Forexample the assay described in J Sanchez-Lopez, et al, “Underivativespolyamine analysis is plant samples by ion pair liquid chromatographycoupled with electrospray tandem mass spectrometry,” Plant Physiologyand Biochemistry, 47 (2009): 592-598, avail online 28 Feb. 2009; M RHakkinen et al, “Analysis of underivatized polyamines by reversed phaseliquid chromatography with electrospray tandem mass spectrometry”, JPharm Biomec Analysis, 44 (2007): 625-634, quantitative isotope dilutionliquid chromatography (LC)/mass spectrometry (MS) assay. Other suitableassays may be used.

In some embodiments, efficacy of a therapeutic described herein isdetermined by assessing neurocognition at week 52 post-dose in pediatricsubjects with MPS II who have an early-stage neurocognitive deficit. Insome embodiments, efficacy of a therapeutic described herein isdetermined by assessing the relationship of CSF glycosaminoglycans (GAG)to neurocognition in an MPS II patient. In some embodiments, efficacy ofa therapeutic described herein is determined by evaluating the effect ofthe therapeutic on physical changes to the CNS in an MPS II patient asmeasured by magnetic resonance imaging (MRI), e.g., volumetric analysisof gray and white matter and CSF ventricles. In some embodiments,efficacy of a therapeutic described herein is determined by evaluatingthe pharmacodynamic effect of the therapeutic on biomarkers, (e.g., GAG,HS) in cerebrospinal fluid (CSF), serum, and urine of an MPS II patient.In some embodiments, efficacy of a therapeutic described herein isdetermined by evaluating the impact of the therapeutic on quality oflife (QOL) of an MPS II patient. In some embodiments, efficacy of atherapeutic described herein is determined by evaluating the impact ofthe therapeutic on motor function of an MPS II patient. In someembodiments, efficacy of a therapeutic described herein is determined byevaluating the effect of the therapeutic on growth and on developmentalmilestones of an MPS II patient.

As expressed from the rAAV vector described herein, expression levels ofhIDS of at least about 2% as detected in the CSF, serum, or othertissue, may provide therapeutic effect. However, higher expressionlevels may be achieved. Such expression levels may be from 2% to about100% of normal functional human IDS levels. In certain embodiments,higher than normal expression levels may be detected in CSF, serum, orother tissue.

In certain embodiments, the methods of treating, preventing, and/orameliorating MPS II and/or symptoms thereof described herein result in asignificant increase in neurocognitive developmental quotient (DQ) intreated patients, as assessed using Bayley Scales of Infant Development.In certain embodiments, the methods of treating, preventing, and/orameliorating MPS II and/or symptoms thereof described herein result in adecline of DQ of no more than 15 points in treated patients relative tountreated/natural history control data in patients with Hunter Syndrome.

In certain embodiments, the methods of treating, preventing, and/orameliorating MPS II and/or symptoms thereof described herein result in asignificant increase in functional human IDS levels. In certainembodiments, the methods of treating, preventing, and/or amelioratingMPS II and/or symptoms thereof described herein result in a significantdecrease in GAG levels, as measured in a sample of a patient's serum,urine and/or cerebrospinal fluid (CSF).

5.3. Combination Therapies

Combinations of gene therapy delivery of the rAAV.hIDS to the CNSaccompanied by systemic delivery of hIDS are encompassed by certainembodiments of the invention. Systemic delivery can be accomplishedusing ERT (e.g., using Elaprase®), or additional gene therapy using anrAAV.hIDS with tropism for the liver (e.g., an rAAV.hIDS bearing an AAV8capsid).

In certain embodiments, an intrathecal administration of rAAV9.hIDS isbe co-administered with a second AAV.hIDS injection, e.g., directed tothe liver. In such an instance, the vectors may be same. For example,the vectors may have the same capsid and/or the same vector genomicsequences. Alternatively, the vector may be different. For example, eachof the vector stocks may designed with different regulatory sequences(e.g., each with a different tissue-specific promoter), e.g., aliver-specific promoter and a CNS-specific promoter. Additionally, oralternatively, each of the vector stocks may have different capsids. Forexample, a vector stock to be directed to the liver may have a capsidselected from AAV8, AAVrh64R1, AAVrh64R2, rh8, rh10, AAV3B, or AAVdj,among others. In such a regimen, the doses of each vector stock may beadjusted so that the total vector delivered intrathecally is within therange of about 1×10⁸ GC to x 1×10¹⁴ GC; in other embodiments, thecombined vector delivered by both routes is in the range of 1×10¹¹ GC to1×10¹⁶ GC. Alternatively, each vector may be delivered in an amount ofabout 10⁸ GC to about 10¹² GC/vector. Such doses may be deliveredsubstantially simultaneously, or at different times, e.g., from about 1day to about 12 weeks apart, or about 3 days to about 30 days, or othersuitable times.

In some embodiments a method for treatment comprises: (a) dosing apatient having MPS II and/or the symptoms Hunter syndrome with asufficient amount of hIDS enzyme or liver-directed rAAV-hIDUA to inducetransgene-specific tolerance; and (b) administering an rAAV.hIDS to thepatient's CNS, which rAAV.hIDS directs expression of therapeutic levelsof hIDS in the patient.

In further embodiments, a method of treating a human patient having MPSII and/or the symptoms associated with Hunter syndrome is provided whichinvolves tolerizing a patient having MPS II and/or the symptomsassociated Hunter syndrome with a sufficient amount of hIDS enzyme orliver-directed rAAV-hIDS to induce transgene-specific tolerance,followed by rAAV-mediated delivery of hIDS to the patient. In certainembodiments, the patient is administered an rAAV.hIDS via liver-directedinjections e.g., when the patient is less than 4 weeks old (neonatalstage) or an infant, in order to tolerize the patient to hIDS, and thepatient is subsequently administered rAAV.hIDS via intrathecalinjections when the patient is an infant, child, and/or adult to expresstherapeutic concentrations of hIDS in the CNS.

In one example, the MPS II patient is tolerized by delivering hIDS tothe patient within about two weeks of birth, e.g., within about 0 toabout 14 days, or about 1 day to 12 days, or about day 3 to about day10, or about day 5 to about day 8, i.e., the patient is a newborninfant. In other embodiments, older infants may be selected. Thetolerizing dose of hIDS may be delivered via rAAV. However, in anotherembodiment, the dose is delivered by direct delivery of the enzyme(enzyme replacement therapy). Methods of producing recombinant hIDS havebeen described in the literature.

Additionally, a recombinant hIDS commercially produced as Elaprase®(idursulfase) may be useful for systemic delivery. Although currentlyless preferred, the enzyme may be delivered via “naked” DNA, RNA, oranother suitable vector. In one embodiment, the enzyme is delivered tothe patient intravenously and/or intrathecally. In another embodiment,another route of administration is used (e.g., intramuscular,subcutaneous, etc). In one embodiment, the MPS II patient selected fortolerizing is incapable of expressing any detectable amounts of hIDSprior to initiation of the tolerizing dose. When recombinant human IDSenzyme is delivered, intravenous rhIDS injections may consist of about0.5 mg/kg body weight. Alternatively, a higher or lower dose isselected. Similarly, when expressed from a vector, lower expressedprotein levels may be delivered. In one embodiment, the amount of hIDSdelivered for tolerizing is lower than a therapeutically effectiveamount. However, other doses may be selected.

Typically, following administration of the tolerizing dose, thetherapeutic dose is delivered to the subject, e.g., within about threedays to about 6 months post-tolerizing dose, more preferably, about 7days to about 1 month post-tolerizing dose. However, other time pointswithin these ranges may be selected, as may longer or shorter waitingperiods.

As an alternative, immunosuppressive therapy may be given in addition tothe vector—before, during and/or subsequent to vector administration.Immunosuppressive therapy can include prednisolone, mycophenolatemofetil (MMF) and tacrolimus or sirolimus as described supra. Atacrolimus-free regimen described infra may be preferred.”

5.4. Manufacture

One embodiment provides for the manufacture of the rAAV.hIDSpharmaceutical compositions described herein (Example 4, infra). Anillustrative manufacturing process is provided in FIGS. 10A and 10B. TherAAV.hIDS vector can be manufactured as shown in the flow diagram shownin FIGS. 10A and 10B. Briefly, cells are manufactured in a suitable cellculture (e.g., HEK 293) cells. Methods for manufacturing the genetherapy vectors described herein include methods well known in the artsuch as generation of plasmid DNA used for production of the genetherapy vectors, generation of the vectors, and purification of thevectors. In some embodiments, the gene therapy vector is an AAV vectorand the plasmids generated are an AAV cis-plasmid encoding the AAVgenome and the gene of interest, an AAV trans-plasmid containing AAV repand cap genes, and an adenovirus helper plasmid. The vector generationprocess can include method steps such as initiation of cell culture,passage of cells, seeding of cells, transfection of cells with theplasmid DNA, post-transfection medium exchange to serum free medium, andthe harvest of vector-containing cells and culture media. The harvestedvector-containing cells and culture media are referred to herein ascrude cell harvest.

The crude cell harvest may thereafter be subject method steps such asconcentration of the vector harvest, diafiltration of the vectorharvest, microfluidization of the vector harvest, nuclease digestion ofthe vector harvest, filtration of microfluidized intermediate, crudepurification by chromatography, crude purification byultracentrifugation, buffer exchange by tangential flow filtration,and/or formulation and filtration to prepare bulk vector.

A two-step affinity chromatography purification at high saltconcentration followed by anion exchange resin chromatography are usedto purify the vector drug product and to remove empty capsids. Thesemethods are described in more detail in International Patent ApplicationNo. PCT/US2016/065970, filed Dec. 9, 2016 and its priority documents,U.S. Patent Application Nos. 62/322,071, filed Apr. 13, 2016 and62/226,357, filed Dec. 11, 2015 and entitled “Scalable PurificationMethod for AAV9”, which is incorporated by reference herein.Purification methods for AAV8, International Patent Application No.PCT/US2016/065976, filed Dec. 9, 2016 and is priority documents U.S.Patent Application Nos. 62/322,098, filed Apr. 13, 2016 and 62/266,341,filed Dec. 11, 2015, and rh10, International Patent Application No.PCT/US16/66013, filed Dec. 9, 2016 and its priority documents, U.S.Patent Application No. 62/322,055, filed Apr. 13, 2016 and 62/266,347,entitled “Scalable Purification Method for AAVrh10”, also filed Dec. 11,2015, and for AAV1, International Patent Application No.PCT/US2016/065974, filed Dec. 9, 2016 and its priority documents U.S.Patent Application Nos. 62/322,083, filed Apr. 13, 2016 and 62/26,351,for “Scalable Purification Method for AAV1”, filed Dec. 11, 2015, areall incorporated by reference herein.

5.5. Apparatus and Method for Delivery of A Pharmaceutical Compositioninto Cerebrospinal Fluid

In one aspect, the vectors provided herein may be administeredintrathecally via the method and/or the device provided in this sectionand described further in the Examples and FIG. 11. Alternatively, otherdevices and methods may be selected. The method comprises the steps ofadvancing a spinal needle into the cisterna magna of a patient,connecting a length of flexible tubing to a proximal hub of the spinalneedle and an output port of a valve to a proximal end of the flexibletubing, and after said advancing and connecting steps and afterpermitting the tubing to be self-primed with the patient's cerebrospinalfluid, connecting a first vessel containing an amount of isotonicsolution to a flush inlet port of the valve and thereafter connecting asecond vessel containing an amount of a pharmaceutical composition to avector inlet port of the valve. After connecting the first and secondvessels to the valve, a path for fluid flow is opened between the vectorinlet port and the outlet port of the valve and the pharmaceuticalcomposition is injected into the patient through the spinal needle, andafter injecting the pharmaceutical composition, a path for fluid flow isopened through the flush inlet port and the outlet port of the valve andthe isotonic solution is injected into the spinal needle to flush thepharmaceutical composition into the patient.

In another aspect, a device for intracisternal delivery of apharmaceutical composition is provided. The device includes a firstvessel containing an amount of a pharmaceutical composition, a secondvessel containing an isotonic solution, and a spinal needle throughwhich the pharmaceutical composition may be ejected from the devicedirectly into cerebrospinal fluid within the cisterna magna of apatient. The device further includes a valve having a first inlet portinterconnected to the first vessel, a second inlet port interconnectedto the second vessel, an outlet port interconnected to the spinalneedle, and a luer lock for controlling flow of the pharmaceuticalcomposition and isotonic solution through the spinal needle.

As used herein, the term Computed Tomography (CT) refers to radiographyin which a three-dimensional image of a body structure is constructed bycomputer from a series of plane cross-sectional images made along anaxis.

The apparatus or medical device 10 as shown in FIG. 11 includes one ormore vessels, 12 and 14, interconnected via a valve 16. The vessels, 12and 14, provide a fresh source of a pharmaceutical composition, drug,vector, or like substance and a fresh source of an isotonic solutionsuch as saline, respectively. The vessels, 12 and 14, may be any form ofmedical device that enables injection of fluids into a patient.

By way of example, each vessel, 12 and 14, may be provided in the formof a syringe, cannula, or the like. For instance, in the illustratedembodiment, the vessel 12 is provided as a separate syringe containingan amount of a pharmaceutical composition and is referred to herein as a“vector syringe”. Merely for purposes of example, the vessel 12 maycontain about 10 cc of a pharmaceutical composition or the like.

Likewise, the vessel 14 may be provided in the form of a separatesyringe, cannula, or the like that contains an amount of saline solutionand may be referred to as a “flush syringe”. Merely for purposes ofexample, the vessel 14 may contain about 10 cc of a saline solution.

As an alternative, the vessels 12 and 14 may be provided in forms otherthan syringes and may be integrated into a single device, such as anintegrated medical injection device have a pair of separate chambers,one for the pharmaceutical composition and one for saline solution.Also, the size of the chambers or vessels may be provided as needed tocontain a desired amount of fluid.

In the illustrated embodiment, the valve 16 is provided as a 4-waystopcock having a swivel male luer lock 18. The valve 16 interconnectsthe vessels 12 and 14 (i.e., the vector syringe and flush syringe in theillustrated embodiment), and the swivel male luer lock enables a paththrough the valve 16 to be closed or opened to each of the vessels 12and 14. In this way, the path through the valve 16 may be closed to boththe vector syringe and flush syringe or may be open to a selected one ofthe vector syringe and flush syringe. As an alternative to a 4-waystopcock, the valve may be a 3-way stopcock or fluid control device.

In the illustrated embodiment, the valve 16 is connected to one end of alength of extension tubing 20 or the like conduit for fluid. The tubing20 may be selected based on a desired length or internal volume. Merelyby way of example, the tubing may be about 6 to 7 inches in length.

In the illustrated embodiment, an opposite end 22 of the tubing 12 isconnected to a T-connector extension set 24 which, in turn, is connectedto a spinal needle 26. By way of example, the needle 26 may be a fiveinch, 22 or 25 gauge spinal needle. In addition, as an option, thespinal needle 26 may be connected to an introducer needle 28, such as athree and a half inch, 18 gauge introducer needle.

In use, the spinal needle 26 and/or optional introducer needle 28 may beadvanced into a patient towards the cisterna magna. After needleadvancement, Computed Tomography (CT) images may be obtained that permitvisualization of the needle 26 and/or 28 and relevant soft tissues(e.g., paraspinal muscles, bone, brainstem, and spinal cord). Correctneedle placement is confirmed by observation of Cerebrospinal Fluid(CSF) in the needle hub and visualization of a needle tip within thecisterna magna. Thereafter, the relatively short extension tubing 20 maybe attached to the inserted spinal needle 26, and the 4-way stopcock 16may then be attached to the opposite end of the tubing 20.

The above assembly is permitted to become “self-primed” with thepatient's CSF. Thereafter, the prefilled normal saline flush syringe 14is attached to a flush inlet port of the 4-way stopcock 16 and then thevector syringe 12 containing a pharmaceutical composition is attached toa vector inlet port of the 4-way stopcock 16. Thereafter, the outputport of the stopcock 16 is opened to the vector syringe 12, and thecontents of the vector syringe may be slowly injected through the valve16 and assembled apparatus and into the patient over a period of time.Merely for purposes of example, this period of time may be approximately1-2 minutes and/or any other time of desire.

After the contents of the vector syringe 12 are injected, the swivellock 18 on the stopcock 16 is turned to a second position so that thestopcock 16 and needle assembly can be flushed with a desired amount ofnormal saline using the attached prefilled flush syringe 14. Merely byway of example, 1 to 2 cc of normal saline may be used; although greateror lesser amounts may be used as needed. The normal saline ensures thatall or most of the pharmaceutical composition is forced to be injectedthrough the assembled device and into the patient and so that little ornone of the pharmaceutical composition remains in the assembled device.

After the assembled device has been flushed with the saline, theassembled device in its entirely, including the needle(s), extensiontubing, stopcock, and syringes are slowly removed from the subject andplaced onto a surgical tray for discarding into a biohazard wastereceptacle or hard container (for the needle(s)).

A screening process may be undertaken by a principal investigator whichmay ultimately lead to an intracisternal (IC) procedure. The principalinvestigator may describe the process, procedure, the administrationprocedure itself, and all potential safety risks in order for thesubject (or designated caregiver) to be fully informed. Medical history,concomitant medications, physical exam, vital signs, electrocardiogram(ECG), and laboratory testing results are obtained or performed andprovided to a neuroradiologist, neurosurgeon, and anesthesiologist foruse in screening assessment of subject eligibility for the IC procedure.

To allow adequate time to review eligibility, the following proceduresmay be performed at any time between the first screening visit and up toone week prior to a study visit. For example, on “Day 0”, Head/NeckMagnetic Resonance Imaging (MRI) with and without gadolinium (i.e.,eGFR>30 mL/min/1.73 m²) may be obtained. In addition to the Head/NeckMRI, the investigator may determine the need for any further evaluationof the neck via flexion/extension studies. The MRI protocol may includeT1, T2, DTI, FLAIR, and CINE protocol images.

In addition, Head/Neck MRA/MRV may be obtained as per institutionalprotocol (i.e., subjects with a history of intra/transdural operationsmay be excluded or may need further testing (e.g., radionucleotidecisternography)) that allows for adequate evaluation of CSF flow andidentification of possible blockage or lack of communication between CSFspaces.

The neuroradiologist, neurosurgeon, and anesthesiologist ultimatelydiscuss and determine the eligibility of each subject for the ICprocedures based on all available information (scans, medical history,physical exam, labs, etc.). An Anesthesia pre-op evaluation may also beobtained from “Day −28” to “Day 1” that provides a detailed assessmentof airway, neck (shortened/thickened) and head range-of-motion (degreeof neck flexion), keeping in mind the special physiologic needs of a MPSsubject.

Prior to an IC procedure, the CT Suite will confirm the followingequipment and medications are present:

Adult lumbar puncture (LP) kit (supplied per institution);

BD (Becton Dickinson) 22 or 25 gauge×3-7″ spinal needle (Quincke bevel);

Coaxial introducer needle, used at the discretion of theinterventionalist (for introduction of spinal needle);

4 way small bore stopcock with swivel (Spin) male luer lock;

T-connector extension set (tubing) with female luer lock adapter,approximate length of 6.7 inches;

Omnipaque 180 (iohexol), for intrathecal administration;

Iodinated contrast for intravenous (IV) administration;

1% lidocaine solution for injection (if not supplied in adult LP kit);

Prefilled 10 cc normal saline (sterile) flush syringe;

Radiopaque marker(s);

Surgical prep equipment/shaving razor;

Pillows/supports to allow proper positioning of intubated subject;

Endotracheal intubation equipment, general anesthesia machine andmechanical ventilator;

Intraoperative neurophysiological monitoring (IONM) equipment (andrequired personnel); and

10 cc syringe containing vector; prepared and transported toCT/Operating Room (OR) suite in accordance with separate PharmacyManual.

Informed Consent for the procedure are confirmed and documented withinthe medical record and/or study file. Separate consent for the procedurefrom radiology and anesthesiology staff is obtained as per institutionalrequirements. Subject has intravenous access placed within theappropriate hospital care unit according to institutional guidelines(e.g., two IV access sites). Intravenous fluids are administered at thediscretion of the anesthesiologist. At the discretion of theanesthesiologist and per institutional guidelines, subject may beinduced and undergo endotracheal intubation with administration ofgeneral anesthesia in an appropriate patient care unit, holding area orthe surgical/CT procedure suite.

A lumbar puncture is performed, first to remove 5 cc of cerebrospinalfluid (CSF) and subsequently to inject contrast (Omnipaque 180)intrathecally to aid visualization of the cisterna magna. Appropriatesubject positioning maneuvers may be performed to facilitate diffusionof contrast into the cisterna magna.

Intraoperative neurophysiological monitoring (IONM) equipment isattached to the subject. Subject is placed onto the CT scanner table inthe prone or lateral decubitus position. Adequate staff must be presentto assure subject safety during transport and positioning. If deemedappropriate, subject may be positioned in a manner that provides neckflexion to the degree determined to be safe during pre-operativeevaluation and with normal neurophysiologic monitor signals documentedafter positioning.

The following staff may be confirmed to be present and identifiedon-site: Interventionalist/neurosurgeon performing the procedure;Anesthesiologist and respiratory technician(s); Nurses and physicianassistants; CT (or OR) technicians; Neurophysiology technician; and SiteCoordinator. A “time-out” may be completed per Joint Commission/hospitalprotocol to verify correct subject, procedure, site, positioning, andpresence of all necessary equipment in the room. The lead siteinvestigator may then confirm with staff that he/she may proceed withprepping the subject.

The subject's skin under the skull base is shaved as appropriate. CTscout images are performed, followed by a pre-procedure planning CT withIV contrast, if deemed necessary by the interventionalist to localizethe target location and to image vasculature. After the target site(cisterna magna) is identified and needle trajectory planned, the skinis prepped and draped using sterile technique as per institutionalguidelines. A radiopaque marker is placed on the target skin location asindicated by the interventionalist. The skin under the marker isanesthetized via infiltration with 1% lidocaine. A 22 G or 25 G spinalneedle is than advanced towards the cisterna magna, with the option touse a coaxial introducer needle.

After needle advancement, CT images are obtained using the thinnest CTslice thickness feasible using institutional equipment (ideally ≤2.5mm). Serial CT images using the lowest radiation dose possible thatallows for adequate visualization of the needle and relevant softtissues (e.g., paraspinal muscles, bone, brainstem, and spinal cord) areobtained. Correct needle placement is confirmed by observation of CSF inthe needle hub and visualization of needle tip within the cisternamagna.

The interventionalist confirms that the vector syringe is positionedclose to, but outside of the sterile field. Prior to handling oradministering the pharmaceutical composition in the vector syringe,gloves, mask, and eye protection are donned by staff assisting theprocedure within the sterile field.

The extension tubing is attached to the inserted spinal needle, which isthen attached to the 4-way stopcock. Once this apparatus is“self-primed” with the subject's CSF, the 10 cc prefilled normal salineflush syringe is attached to a flush inlet port of the 4-way stopcock.The vector syringe is then provided to the interventionalist andattached to a vector inlet port on the 4-way stop cock.

After the outlet port of the stopcock is opened to the vector syringe byplacing the swivel lock of the stopcock in a first position, thecontents of the vector syringe are injected slowly (over approximately1-2 minutes), with care taken not to apply excessive force onto theplunger of the syringe during the injection. After the contents of thevector syringe are injected, the swivel lock of stopcock is turned to asecond position so that the stopcock and needle assembly can be flushedwith 1-2 cc of normal saline using the attached prefilled flush syringe.

When ready, the interventionist then alerts staff that he/she willremove the apparatus from the subject. In a single motion, the needle,extension tubing, stopcock, and syringes are slowly removed from thesubject and placed onto a surgical tray for discarding into a biohazardwaste receptacle or hard container (for the needle).

The needle insertion site is examined for signs of bleeding or CSFleakage and treated as indicated by the investigator. Site is dressedusing gauze, surgical tape and/or Tegaderm dressing, as indicated.Subject is then removed from the CT scanner and placed supine onto astretcher. Adequate staff is present to assure subject safety duringtransport and positioning.

Anesthesia is discontinued and subject cared for following institutionalguidelines for post-anesthesia care. Neurophysiologic monitors areremoved from the subject. The head of the stretcher on which the subjectlies should be slightly raised (˜30 degrees) during recovery. Subject istransported to a suitable post-anesthesia care unit as per institutionalguidelines. After subject has adequately recovered consciousness and isin stable condition, he/she will be admitted to the appropriatefloor/unit for protocol mandated assessments. Neurological assessmentswill be followed as per the protocol and the Primary Investigatoroversees subject care in collaboration with hospital and research staff.

In one embodiment, a method for delivery of a composition providedherein comprises the steps of: advancing a spinal needle into thecisterna magna of a patient; connecting a length of flexible tubing to aproximal hub of the spinal needle and an output port of a valve to aproximal end of the flexible tubing; after said advancing and connectingsteps and after permitting the tubing to be self-primed with thepatient's cerebrospinal fluid, connecting a first vessel containing anamount of isotonic solution to a flush inlet port of the valve andthereafter connecting a second vessel containing an amount of apharmaceutical composition to a vector inlet port of the valve; afterconnecting said first and second vessels to the valve, opening a pathfor fluid flow between the vector inlet port and the outlet port of thevalve and injecting the pharmaceutical composition into the patientthrough the spinal needle; and after injecting the pharmaceuticalcomposition, opening a path for fluid flow through the flush inlet portand the outlet port of the valve and injecting the isotonic solutioninto the spinal needle to flush the pharmaceutical composition into thepatient. In certain embodiment, the method further comprises confirmingproper placement of a distal tip of the spinal needle within thecisterna magna before connecting the tubing and valve to the hub of thespinal needle. In certain embodiments, the confirming step includesvisualizing the distal tip of the spinal needle within the cisternamagna with Computed Tomography (CT) imaging. In certain embodiments, theconfirming step includes observing the presence of the patient'scerebrospinal fluid in the hub of the spinal needle.

In the above-described method, the valve may be a stopcock with a swivelluer lock adapted to swivel to a first position permitting flow from thevector inlet port to the outlet port while simultaneously blocking flowthrough the flush inlet port and to a second position permitting flowfrom the flush inlet port to the outlet port while simultaneouslyblocking flow through the vector inlet port, and wherein the swivel luerlock is positioned into said first position when said pharmaceuticalcomposition is injected the patient and is positioned into said secondposition when said pharmaceutical composition is being flushed into saidpatient by the isotonic solution. In certain embodiments, afterinjecting the isotonic solution into the spinal needle to flush thepharmaceutical composition into the patient, the spinal needle iswithdrawn from the patient with the tubing, valve, and first and secondvessels connected thereto as an assembly. In certain embodiments, thevalve is a 4-way stopcock with a swivel male luer lock. In certainembodiments, the first and second vessels are separate syringes. Incertain embodiments, a T-connector is located at the hub of the spinalneedle and interconnects the tubing to the spinal needle. Optionally,the spinal needle includes an introducer needle at the distal end of thespinal needle. The spinal needle may be a five inch, 22 or 24 gaugespinal needle. In certain embodiments, the introducer needle is a 3.5inch, 18 gauge introducer needle.

In certain aspects, the method utilizes a device which is composed of,at a minimum, a first vessel for containing an amount of apharmaceutical composition; a second vessel for containing an isotonicsolution; a spinal needle through which the pharmaceutical compositionmay be ejected from the device directly into cerebrospinal fluid withinthe cisterna magna of a patient; and a valve having a first inlet portinterconnected to the first vessel, a second inlet port interconnectedto the second vessel, an outlet port interconnected to the spinalneedle, and a luer lock for controlling flow of the pharmaceuticalcomposition and isotonic solution through the spinal needle. In certainembodiments, the valve is a stopcock with a swivel luer lock adapted toswivel to a first position permitting flow from the first inlet port tothe outlet port while simultaneously blocking flow through the secondinlet port and to a second position permitting flow from the secondinlet port to the outlet port while simultaneously blocking flow throughthe first inlet port. Optionally, the valve is a 4-way stopcock with aswivel male luer lock. In certain embodiments, the first and secondvessels are separate syringes. In certain embodiments, the spinal needleis interconnected to the valve via a length of flexible tubing. AT-connector may interconnect the tubing to the spinal needle. In certainembodiments, the spinal needle is a five inch, 22 or 24 gauge spinalneedle. In certain embodiments, the device further comprises anintroducer needle connected to a distal end of the spinal needle.Optionally, the introducer needle is a 3.5 inch, 18 gauge introducerneedle.

This method and this device may each optionally be used for intrathecaldelivery of the compositions provided herein. Alternatively, othermethods and devices may be used for such intrathecal delivery.

The following examples are illustrative only and are not a limitation onthe invention described herein.

6. EXAMPLES Example 1: Protocol for Treatment of Human Subjects

This Example relates to a gene therapy treatment for patients that haveMPS II, i.e., Hunter syndrome. In this example, the gene therapy vector,AAV9.CB.hIDS, a replication deficient adeno-associated viral vector 9(AAV9) expressing a modified hIDS gene encoding the wild-type hIDSenzyme, is administered to the central nervous system (CNS) of the MPSII patients. Doses of the AAV vector are injected directly into the CNSunder general anesthesia. Efficacy of treatment is assessed usingclinical measures of neurocognitive development and/or surrogatemarkers, including biomarkers, e.g., a decrease in pathogenic GAG and/orheparin sulfate (HS) concentration in the subject's CSF or serum, asdescribed herein.

A. Gene Therapy Vector

The gene therapy vector is a non-replicating recombinantadeno-associated virus (AAV) vector of serotype 9 expressing humaniduronate-2-sulfatase (IDS), and is referred to in this Example asAAV9.CB. IDS (see FIG. 1). The AAV9 serotype allows for efficientexpression of the hIDS product in the CNS following IC administration.

The IDS expression cassette is flanked by inverted terminal repeats(ITRs) and expression is driven by a hybrid of the cytomegalovirus (CMV)enhancer and the chicken beta actin promoter (CB7). The transgeneincludes the chicken beta actin intron and a rabbit beta-globinpolyadenylation (polyA) signal.

The vector is suspended in formulation buffer (Elliots B Solution,0.001% Pluronic F68). The construct was packaged in an AAV9 capsid,purified and titered as previously described in M. Lock et al, HumanGene Ther, 21: 1259-1271 (2010). The manufacturing process is describedin more detail in Example 4 below.

Vector Production:

Vector Production:

A series of vectors were generated. One plasmid contains acodon-optimized IDS sequence (nt 1177 to nt 2829 of SEQ ID NO: 11). Twoothers used a short version of the CB7 promoter (CB6) and expressed asecond protein called SUMF1 [SEQ ID NO: 7 and SEQ ID NO: 10]. The vectorgenome produced, AAV.CB7.CI.hIDSco.RBG, has a sequence of nt 2 to nt3967 of SEQ ID NO: 11 while AAV.CB6.hIDSco.IRES.hSUMF1co, has a sequenceof nt 11 to nt 4964 of SEQ ID NO: 8. The plasmids were constructed bycodon-optimizing and synthesizing the hIDS sequence and the resultingconstruct was then cloned into the plasmid pENN.AAV.CB7.CI.RBG orpENN.AAV.CB6.CI.RBG, an AAV2 ITR-flanked expression cassette containingCB7 or CB6, CI and RBG expression elements to give the said vectorgenome.

Still further plasmids were created containing the native human IDScDNA; these plasmids are termed herein pAAV.CB7.CI.hIDS.RBG andpAAV.CB6.CI.hIDS.IRES.SUMF1.RBG. The vector genomes derived from theseplasmids [nt 2 to nt 3967 of SEQ ID NO: 3 and nt 11 to nt 4964 of SEQ IDNO: 5] is single-stranded DNA genome with AAV2 derived ITRs flanking thehIDS expression cassette. Expression from the transgene cassette isdriven by a CB7 or CB6 promoter, a hybrid between a CMV immediate earlyenhancer (C4) and the chicken beta actin promoter, while transcriptionfrom this promoter is enhanced by the presence of the chicken beta actinintron (CI). The polyA signal for the expression cassette is the RBGpolyA. The plasmids were constructed by synthesizing the hIDS sequenceand the resulting construct was then cloned into the plasmidpENN.AAV.CB7.CI.RBG or pENN.AAV.CB6.CI.RBG, an AAV2 ITR-flankedexpression cassette containing CB7, CI and RBG expression elements togive the said vector genome.

Description of the Sequence Elements:

Inverted terminal repeats (ITR): AAV ITRs (GenBank #NC001401) aresequences that are identical on both ends, but in opposite orientation.The AAV2 ITR sequences function as both the origin of vector DNAreplication and the packaging signal of the vector genome, when AAV andadenovirus helper functions are provided in trans. As such, the ITRsequences represent the only cis sequences required for vector genomereplication and packaging.

CMV immediate early enhancer (382 bp, C4; GenBank #K03104.1). Thiselement is present in the vector genome plasmid.

Chicken beta-actin promoter (282 bp; CB; GenBank #X00182.1) is used todrive high-level hIDS expression.

Chicken beta-actin intron: The 973 bp intron from the chicken beta actingene (GenBank #X00182.1) is present in the vector expression cassette.The intron is transcribed, but removed from the mature messenger RNA(mRNA) by splicing, bringing together the sequences on either side ofit. The presence of an intron in an expression cassette has been shownto facilitate the transport of mRNA from the nucleus to the cytoplasm,thus enhancing the accumulation of the steady level of mRNA fortranslation. This is a common feature in gene vectors intended forincreased level of gene expression. This element is present in bothvector genome and plasmids.

Iduronate-2-sulfatase coding sequence: The hIDS sequence was synthesized[SEQ ID NO: 1]. The encoded protein is 550 amino acids [SEQ ID NO: 2;Genbank NP_000193, UnitProtKB/Swiss-Prot (P22304.1)], described earlierin the specification. See, SEQ ID NOs: 1 and 2. A codon-optimized hIDScoding sequence is shown in nt 3423 to nt 4553 of SEQ ID NO: 8 as wellas nt 1937 to nt 3589 of SEQ ID NO: 11.

Polyadenylation Signal: The 127 bp rabbit beta-globin polyadenylationsignal (GenBank #V00882.1) provides cis sequences for efficientpolyadenylation of the antibody mRNA. This element functions as a signalfor transcriptional termination, a specific cleavage event at the 3′ endof the nascent transcript and addition of a long polyadenyl tail. Thiselement is present in both vector genome and plasmids.

B. Dosing & Route of Administration

Patients receive a single intrathecal/intracisternal dose ofrAAV9.CB7.hIDS which ranges from 1.0×10¹³ to 5.0×10¹⁴ GC (flatdoses)—the equivalent of 2.5×10¹⁰ to 3.6×10¹¹ GC/g brain mass of apatient. Alternatively, the following flat doses are administered topatients of the indicated age group:

Newborns: about 3.8×10¹² to about 1.9×10¹⁴ GC;

3-9 months: about 6×10¹² to about 3×10¹⁴ GC;

9-36 months: about 1×10¹³ to about 5×10¹⁴ GC;

3-12 years: about 1.2×10¹³ to about 6×10¹⁴ GC;

12+ years: about 1.4×10¹³ to about 7.0×10¹⁴ GC;

18+ years (adult): about 1.4×10¹³ to about 7.0×10¹⁴ GC.

In order to ensure that empty capsids are removed from the dose ofrAAV9.CB7.hIDS that is administered to patients, empty capsids areseparated from vector particles by cesium chloride gradientultracentrifugation or by ion exchange chromatography during the vectorpurification process, as discussed herein.

C. Patient Subpopulations

Suitable patients include, male or female subjects in age:

Newborns;

3-9 months of age;

9-36 months of age;

3-12 years of age;

12+ years of age;

18+ years of age (adult).

D. Measuring Clinical Objectives

Primary clinical objectives include preventing and/or optionallyreversing the neurocognitive decline associated with MPS II defects.Clinical objectives may be determined by measuring neurocognition, e.g.,as measured by Bayley Scales of Infant and Toddler Development, ThirdEdition, BSID-III. Other appropriate measures are adaptive behavioralassessments, e.g., as measured by Vineland Adaptive Behavior Scales,Second Edition (VABS-II), and quality of life measure, e.g., as measuredby Infant Toddler Quality of Life Questionnaire™ (ITQOL).

Secondary endpoints include evaluation of biomarkers and clinicaloutcomes. Optionally hearing may be assessed as a secondary endpoint.Urine is evaluated for total GAG content, as well as heparan sulfate.Serum is evaluated for IDS activity, anti-IDS antibodies, GAG, andconcentration of the heparin cofactor II-thrombin complex. CSF isevaluated for GAG, IDS activity, anti-IDS antibodies, and heparinsulfate. The presence of anti-IDS antibodies is assessed, as is thepharmacokinetics of IDS expression in CSF and serum. Volumetric analysisof gray and white matter and CSF ventricles is also performed by MRI.

Example 2: Studies in Murine Models of Mucopolysaccharidosis TypeII—Delivery of an Adeno-Associated Virus Vector into CSF AttenuatesCentral Nervous System Disease in Mucopolysaccharidosis Type II Mice

A. AAV9 Delivery into Cerebrospinal Fluid Corrects CNS Disease

Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storagedisorder typically manifesting in early childhood with bone and jointdeformities, cardiac and respiratory disease, and developmental delay.Systemic delivery of the deficient enzyme, iduronate-2-sulfatase (IDS),improves many symptoms of MPS II, but because the enzyme does not crossthe blood-brain barrier, there is currently no effective method toprevent the progression of central nervous system (CNS) disease. Using amouse model of MPS II, AAV serotype 9 vector-mediated delivery of theIDS gene was evaluated as a means of achieving continuous IDS expressionin the CNS. IDS knockout mice received a single injection into thelateral ventricle of one of three vector doses (low—3×10⁸, mid—3×10⁹ orhigh—3×10¹⁰ genome copies) and were sacrificed either three weeks aftervector administration for assessment of vector biodistribution and IDSexpression (n=7-8 mice per group), or 3 months after vectoradministration to evaluate the impact of gene transfer on diseaseprogression (n=7-8 mice per group). IDS activity was detectable incerebrospinal fluid, reaching 15% of wild-type levels in the low-dosecohort and 268% of normal at the highest dose. Brain enzyme activityranged from 2.7% of normal in the low-dose cohort to 32% in thehigh-dose cohort. Quantification of brain storage lesions by stainingfor the ganglioside GM3 indicated dose-dependent correction, with 35%,46%, and 86% reductions in the low-, mid-, and high-dose cohorts,respectively. Treated mice also demonstrated improved cognitive functionin a novel object recognition test. These findings indicate thatintrathecal AAV-mediated gene transfer serves as a platform forsustained enzyme delivery to the CNS, addressing this critical unmetneed for patients with MPS II.

B. Pharmacologic and Neurobehavioral Effects of AAV9.CB.hIDS

A knockout mouse model (IDS-knockout; IDS^(y/−)) of MPS II was createdby replacing exons 4 and 5 of the IDS gene with the neomycin resistancegene (see Garcia et al., 2007, J Inherit Metab Dis, 30:924-34). Themodel exhibits no detectable enzyme activity and develops histologicalstorage lesions similar to those found in MPS II patients. TheIDS-knockout mouse exhibits many of the clinical features of MPS II,including skeletal abnormalities. The neurobehavioral phenotype of themice has not been extensively evaluated, although some studies haveindicated abnormalities (Muenzer et al., 2001, Acta Paediatr Suppl,91(439): 99-9) and therefore is a relevant model for studying theeffects of AAV9.CB.hIDS as a treatment for MPS II. In fact, this is thesame knockout mouse model which has been used to assess the effect ofenzyme replacement therapy in MPS II in support of clinical trials inthis population. The following study carried out in this MPS II mousemodel established the therapeutic activity AAV9.CB.hIDS.

C. Materials and Methods

Vectors. The human IDUA and IDS cDNAs were cloned into an expressionconstruct containing a chicken beta actin promoter, CMV enhancer,intron, and rabbit beta globin polyadenylation sequence. The expressionconstructs were flanked by AAV2 inverted terminal repeats. AAV9 vectorswere generated from these constructs by triple transfection of HEK 293cells and iodixanol purification as previously described (Lock et al.(2010). Hum Gene Ther 21: 1259-71).

Animal procedures. All animal protocols were approved by theInstitutional Animal Care and Use Committee of the University ofPennsylvania. IDS knockout mice were obtained from Jackson Laboratory(Stock no: 024744) and bred in house. Wild type C57BL/6 males from thecolony served as controls. At 2-3 months of age, animals wereanesthetized with isoflurane and injected ICV with 5 μL vector dilutedin sterile phosphate buffered saline. CSF was collected at the time ofnecropsy by suboccipital puncture with a 32-gauge needle connected topolyethylene tubing. Terminal serum samples were collected by cardiacpuncture. Animals were euthanized by exsanguination underketamine/xylazine anesthesia. Death was confirmed by cervicaldislocation. The brain, heart, lungs, liver and spleen were collected ondry ice. For histology experiments, the brain was divided into ananterior half which was fixed for LIMP2 immunohistochemistry, and aposterior half fixed for GM3 immunohistochemistry. Two cohorts of micewere used for behavior experiments. An initial cohort of wild type andIDS KO mice was tested in a battery of procedures to determine if genedeletion affected learning and memory. A second cohort of mice treatedwith low, medium or high vector doses of AAV9.CB.hIDS (3×10⁸, 3×10⁹, or3×10¹⁰ genome copies (GC) respectively) was used to investigate thefeasibility of IT delivery as strategy to rescue of behavior deficits.

Behavior procedures: All behavior procedures were performed by operatorsblinded to genotype and treatment group.

Open Field Activity: Spontaneous activity in an open field was measuredwith a Photobeam Activity System (PAS)-Open Field (San DiegoInstruments). Mice were individually placed in the arena for a single 10minute trial. Horizontal and vertical beam breaks were collected toassess general locomotion and rearing activity.

Y maze: Short term memory was assessed with a standard Y-shaped maze(San Diego Instruments). The sequence and number of arm entries wasrecorded during an 8 min trial. A spontaneous alternation (SA) wasdefined as sequential entry into all three arms of the maze withoutimmediately returning to a previously entered arm. Total arm entries(AE) were collected as a measure of motor activity. The percentspontaneous alternation was calculated as % SA=(SA/(AE−2)*100).

Contextual fear conditioning: Conditioning experiments were performed asdescribed by Abel et al. Cell. 1997 Mar. 7; 88(5):615-26. On thetraining day, mice were allowed to explore the unique conditioningchamber (Med Associates) for 300 seconds. A non-signaled, 1.5 mAcontinuous footshock was delivered between 248-250 seconds. After anadditional 30 seconds in the chamber, the mice were returned to theirhome cage. Twenty-four hours later, recall of spatial context wasassessed for 5 consecutive minutes in the same chamber where trainingoccurred. Memory was assessed with software used to score freezingbehavior (Freezescan, CleverSystems). The percent freezing in the 2.5minutes prestimulus epoch of the training session is compared to thepercent freezing upon reexposure to the chamber. An increase in freezingindicates that learning has occurred.

Novel object recognition: The experimental apparatus consisted of a greyrectangular arena (60 cm×50 cm×26 cm) on a white floor and two uniqueobjects: 3.8×3.8×15 cm metal bars and 3.2 cm dia.×15 cm PVC pipes. Priorto exposure to the apparatus, mice were handled 1-2 minutes/day for fivedays. During a five day habituation phase, mice were allowed to explorean empty arena for five minutes/day. During the training phase, miceexplored two of the same objects for 15 minutes to establishfamiliarization. In the recall phase 24 hours later, mice were returnedto the arena with one now-familiar object and a novel object. Mice willpreferentially explore the novel object. A reduced preference fornovelty suggests a failure to recall the familiar object and thus alearning deficit. All sessions were recorded, and time spent exploringobjects was scored with an open source image analysis program (Patel elal., Front Behav Neurosci. 2014 Oct. 8; 8:349).

Enzyme and GAG assays. GAG, Hex and IDUA assays were performed aspreviously described (Hinderer et al. (2015). Mol Ther 23: 1298-307).IDS activity was measured by incubating 10 μL sample with 20 μL of 1.25mM 4-methylumbelliferyl α-L-idopyranosiduronic acid 2-sulfate (SantaCruz Biotechnology) dissolved in 0.1 M sodium acetate with 0.01 M leadacetate, pH 5.0. After incubating 2 hours at 37° C., 45 μL of McIlvain'sbuffer (0.4 M sodium phosphate, 0.2 M sodium citrate, pH 4.5) and 5 μLrecombinant human iduronidase (Aldurazyme, 0.58 mg/mL, Genzyme) wereadded to the reaction mixture and incubated overnight at 37° C. Themixture was diluted in glycine buffer, pH 10.9, and released 4-MU wasquantified by fluorescence (excitation 365 nm, emission 450 nm) comparedwith standard dilutions of free 4-MU.

Histology. The brain was divided into an anterior half which was fixedfor LIMP2 immunohistochemistry, and a posterior half fixed for GM3immunohistochemistry. LIMP2 and GM3 immunohistochemistry were performedas previously described (Hinderer et al. (2015). Mol Ther 23: 1298-307).The number of cells staining positive for LIMP2 and GM3 was quantifiedin 4 brain sections from each animal by a blinded reviewer.

Vector biodistribution. Tissues for vector biodistribution analysis werequickly dissected and frozen on dry ice. Samples were stored at −80° C.until the time of analysis. DNA was isolated from tissues using theQIAmp DNA Mini Kit and vector genomes quantified by TaqMan PCR asdescribed (Wang, et al. (2011). Hum. Gene Ther. 22: 1389-1401).

ELISA for anti-hIDS antibodies. Polystyrene ELISA plates were coatedovernight with recombinant human IDS (R&D Systems) 5 μg/mL in PBStitrated to pH 5.8. Plates were washed and blocked 1 hour in 2% bovineserum albumin in neutral PBS. Plates were then incubated with serumsamples diluted 1:1000 in PBS. Bound antibody was detected with HRPconjugated goat anti-mouse antibody (Abcam) diluted 1:10,000 in PBS with2% BSA. The assay was developed using tetramethylbenzidine substrate andstopped with 2 N sulfuric acid before measuring absorbance at 450 nm.Titers were determined from a standard curve generated by serialdilution of a positive serum sample arbitrarily assigned a titer of1:10,000.

Statistics. Tissue GAG content, Hex activity, and brain storage lesionsin treated and untreated mice were compared using a one-way ANOVAfollowed by Dunnett's multiple comparisons test. Open field and Y mazedate were analyzed with Students t-test. A two-way ANOVA and Dunnett'spost-hoc analysis was applied to the fear conditioning data to assesstrial and genotype effects. For the novel object recognition test, timeexploring the novel object vs familiar object was compared using at-test for each group, followed by a Bonferroni correction for multiplecomparisons.

Four age-matched groups of male IDS-knockout mice and one group of wildtype littermates (8 mice per group (n=8)) received the followingtreatments at 2-3 months of age:

-   -   Group 1: Untreated    -   Group 2: Intracerebroventricular AAV9.CB.hIDS Low dose 3×10⁸ GC        (1.875×10⁹ GC/g brain mass)    -   Group 3: Intracerebroventricular AAV9.CB.hIDS Mid dose 3×10⁹ GC        (1.875×10¹⁰ GC/g brain mass)    -   Group 4: Intracerebroventricular AAV9.CB.hIDS High dose 3×10¹⁰        GC (1.875×10¹¹ GC/G brain mass)    -   Group 5: Untreated wild type littermates

D. Results:

Male IDS^(y/−) mice between 2 and 3 months of age were treated with anintracerebroventricular (ICV) injection of an AAV9 vector expressinghuman IDS from a chicken beta actin promoter with a cytomegalovirusenhancer (AAV9.CB.hIDS). In an initial cohort, mice were treated withone of three vector doses [3×10⁸, 3×10⁹, or 3×10¹⁰ genome copies (GC)]and sacrificed 3 weeks after vector administration for assessment ofvector biodistribution and IDS expression. Untreated IDS^(y/−) mice andwild type male littermates served as controls. Tissues were harvestedfor biodistribution analysis. Analysis of vector biodistribution in thehigh-dose group demonstrated efficient brain targeting, with an averageof one vector genome per host diploid genome (FIG. 3). Consistent withprevious studies of AAV delivery into CSF, there was also vector escapeto the periphery and efficient hepatic targeting, with more than onevector genome per host diploid genome (FIG. 3). Brain tissue, CSF, andserum all exhibited dose-dependent increases in IDS activity, whichapproached or exceeded wild type levels in the high-dose group (FIGS.2A-2C). Antibodies to human IDS were detected in serum of severalanimals in the mid- and high-dose cohorts, although this did not appearto significantly affect circulating enzyme activity (FIG. 8).

In order to evaluate the therapeutic potential of IT AAV9-mediateddelivery of the IDS gene, an additional cohort of IDS^(y/−) mice wastreated with equivalent vector doses, then evaluated at later timepoints to assess the impact of gene transfer on disease progression. Twomonths after vector administration the mice were subjected to behavioraland neurocognitive tests. Three months after treatment the animals weresacrificed and tissues harvested for histological and biochemicalassessment of disease activity.

Consistent with high levels of serum enzyme activity, GAG storage wasreduced in the liver, and there was a nonsignificant trend towardnormalization of GAG storage in the heart (FIGS. 4A-4B). In addition,activity of the lysosomal enzyme hexosaminidase (Hex), which isupregulated in the setting of GAG storage, was normalized in bothtissues (FIGS. 4C-4D). These data indicate potential for systemictherapeutic activity after intrathecal administration of AAV9.CB.hIDS.

The brains of untreated MPS II mice showed clear histological evidenceof lysosomal storage in neurons, including accumulation of the lysosomalmembrane protein LIMP2, as well as secondary storage of gangliosidesincluding GM3 (FIG. 5L). Treated mice demonstrated a dose-dependentdecrease in neuronal storage lesions evident by both LIMP2 and GM3staining (FIGS. 5A-5L). Based on this data the low dose of 1.875×10⁹GC/g was estimated to be the Minimum Effective Dose (MED) in mice, asthis was the lowest dose at which mice exhibited a significant(approximately 50%) reduction in both GM3 and LIMP2 storage lesions.

Behavioral testing was performed when the mice reached 4-5 months ofage, 2 months after vector administration. A comprehensive battery oftests was performed to evaluate general behavior as well as short andlong-term memory. IDS^(y/−) mice showed normal exploratory activity inan open field arena (FIGS. 9A-9C). Spontaneous alternations in a Y-maze(FIG. 9D) were used to assess short term working memory. IDS^(y/−) micehad similar numbers of arm entries and equivalent spontaneousalternations to wild type littermates, demonstrating intact short termmemory. Long term memory was assessed using classic contextual fearconditioning (FC) and novel object recognition (NOR). In FC, theassociation of an aversive stimulus with a specific context evokesfreezing behavior upon reexposure to that context. All mice showed anincrease in the percent time freezing during the recall phase of thetest demonstrating that learning occurred, however IDS^(y/−) mice showedreduced freezing relative to wild type littermates (FIG. 6A). In treatedanimals, there was no clear improvement in contextual fear conditioning,although treatment effects were difficult to evaluate due to the smalldifference between normal and untreated IDS^(y/−) mice (FIG. 6B). InNOR, mice are allowed to explore a pair of similar objects. Twenty fourhours after training, one now-familiar object is replaced with a novelobject. Mice have an innate propensity to explore the novel object;failure to do so demonstrates a lack of recognition of the familiarobject and reveals a memory deficit. Wild type mice demonstrated apreference for a novel object, but IDS^(y/−) mice did not show apreference. Remarkably, intrathecal AAV9 gene therapy rescued the longterm NOR deficits observed in the IDS^(y/−) mice (FIG. 6C). Objectdiscrimination appeared to be improved in all treated IDS^(y/−) mice,with all groups exhibiting a trend toward greater percentage timeexploring a novel object compared with a familiar one. The preferencefor the novel object was statistically significant only in the mid-dosecohort, although the study was not sufficiently powered to compare therelative degree of rescue of behavioral deficits among dosing groups(FIGS. 6A-6C).

One drawback of evaluating IT AAV delivery in a murine disease model isthat the extremely small mouse CNS—with a brain mass of just 0.4 g andCSF volume of 40 μL—may not accurately model the diffusion of vector andsecreted enzyme in the human brain, which is 3,500-fold larger. Inprevious studies of the related lysosomal storage diseasemucopolysaccharidiosis Type I (MPSI), we utilized naturally occurringlarge animal disease models to address this issue. [Hinderer et al, MolTherapy: the Journal of the Am Soc Gen Therapy, 2014: 22: 2018-2027;Hinderer et al, Mol Therapy: the Journal of the Am Soc Gen Therapy,2015: 23: 1298-1307]. With average brain masses of 30 g and 72 g,respectively, the MPS I cat and dog provided much more realistic modelsto address the challenge of achieving widespread vector and enzymedelivery in the human brain. Studies carried out in these modelsprovided critical evidence of the efficacy of IT AAV9 delivery for MPS I[Hinderer et al, Mol Therapy: the Journal of the Am Soc Gen Therapy,2014: 22: 2018-2027; Hinderer et al, Mol Therapy: the Journal of the AmSoc Gen Therapy, 2015: 23: 1298-1307]. In order to determine whethersimilar efficacy would be possible using this same approach in MPS II,we performed a parallel dose-ranging study in MPS I mice to examine therelative efficiency of enzyme expression and cross-correction in MPS II.MPS I mice were treated with a vector identical to that used forIDS^(y/−) mice, with the exception of the α-L-iduronidase (IDUA)transgene in place of IDS. As with the MPS II study, MPS I mice weretreated with doses of 3×10⁸, 3×10⁹, or 3×10¹⁰ GC (n=8 per group) at 2-3months of age and sacrificed at day 21 post-treatment for measurement ofbrain enzyme activity or at 3 months post-treatment for histologicalanalysis. There was a similar dose response in brain enzyme expressionto that observed in IDS^(y/−) mice, although expression of IDUA appearedsomewhat more efficient compared with IDS, both in absolute enzymeactivity and relative to wild type expression levels (FIG. 7A).Correction of brain storage lesions was similar between the two diseasemodels, although treatment appeared modestly more effective in MPS Imice at the lowest vector dose (FIGS. 7A-7B). Together these resultssuggest that IT AAV9-mediated gene transfer for MPS II yields correctionof brain storage lesions nearly as efficient as that observed in MPS I,and that this approach remains effective when scaled up from mice.However, slightly higher vector doses compared to MPS I may be necessaryin MPS II in order to overcome less efficient enzyme expression in thebrain.

In the present study, IT delivery of an AAV9 vector carrying the humanIDS gene in MPS II mice resulted in therapeutic levels of expression inthe CNS and resolution of brain storage lesions. Functional improvementwas indicated by greater novel object discrimination in treated micecompared with untreated controls. Neurocognitive deficits have not beenpreviously well characterized in IDS^(y/−) mice. These findingsdemonstrate that IDS^(y/−) mice have normal exploratory activity in theopen field as well as similar arm entries in the Y-maze relative to wildtype littermates. These locomotor assessments are critical inconsidering other types of behavior that require normal exploration. TheIDS^(y/−) mice have been found to have intact short term working memorydemonstrated by similar spontaneous alternations in the Y-maze comparedto controls. In contrast, Higuchi et al. [Mol Genet and Metabolism,2012: 107: 122-128] reported reduced spontaneous alternations andincreased arm entries in 32 week old IDS^(y/−) mice. The differingY-maze results may be due to the different age at testing and underscorethe progressive nature of MPS II. We assessed the impact of IDSdeficiency on two forms of long term memory. A mild deficit wasidentified in contextual fear conditioning in the IDS^(y/−) mouse.Although IDS^(y/−) mice were able to recall the context in which theyreceived an aversive stimulus, they showed a significantly reducedfreezing response compared to wild type littermates. IDS^(y/−) mice thatreceived IT delivery of AAV9 showed freezing responses similar touninjected mice. Therefore, no recovery was detected. A long term memorydeficit was also found in novel object recognition. In NOR, IDS^(y/−)mice showed no preference for a novel object relative to a familiarobject. Vector treated mice demonstrated recovery of the NOR deficitseen in untreated IDS^(y/−) mice. Object discrimination wasstatistically significant only in the mid-dose cohort, although with 8animals per group, the study was not well powered to evaluate the doseresponse of behavioral endpoints. The ability of IT delivery of AAV9 todifferentially affect recovery of two types of long term memory may bedue to different neural substrates required for each task. Contextualfear conditioning is a hippocampal-dependent task as opposed to NORwhich requires the perienteorhinal cortex [Oliveira, et al,Post-training reversible inactivation of the hippocampus enhances novelobject recognition memory. Learning & memory (Cold Spring Harbor, N.Y.)2010; 17:155-160; Abel, et al, Cell 1997; 88:615-626].

Comparison of the efficiency of IT AAV9 delivery in mouse models of MPSII and MPS I indicated that enzyme expression and tissue correction weresimilar between the two diseases, providing evidence that IT AAVdelivery for MPS II is efficient enough to yield widespread diseasecorrection in the context of much larger brain sizes and CSF volumes, ashas previously been shown in large animal models of MPS I [Hinderer etal, Mol Therapy: the Journal of the Am Soc Gen Therapy, 2014: 22:2018-2027; Hinderer et al, Mol Therapy: the Journal of the Am Soc GenTherapy, 2015: 23: 1298-1307]. Interestingly, expression of thedeficient enzyme was somewhat less efficient in MPS II compared with MPSI. This could simply be a product of the expression efficiency of theseparticular vectors, although the control elements in the expressionconstructs were identical. Some studies have suggested that expressionof sulfatases such as IDS can be limited by the availability of thesulfatase modifying factor, SUMF1, which is required forpost-translational modification of IDS [Fraldi, et al, Biochemical J,2007: 403: 305-312. However, pilot experiments evaluating co-expressionof IDS and SUMF1 have not demonstrated more active IDS expression (datanot shown). Regardless, correction of storage lesions was nearly asefficient in MPS II mice as in MPS I mice, indicating that gene transfershould be similarly effective for both MPS I and MPS II patients, thoughin the latter case modestly higher vector doses may be necessary foroptimal outcomes.

In addition to CNS gene transfer, there was significant livertransduction and peripheral enzyme expression in treated MPS II mice,indicating possible systemic benefits of IT AAV9 delivery in MPS II.This is consistent with studies of IT AAV delivery in a variety of otherspecies [Passini et al, Hu Gene Therapy, 2014; 25: 619-630; Hinderer etal, Molecular Therapy—Methods & Clinical Development 2014; 1; Hindereret al, Molecular therapy: the journal of the American Society of GeneTherapy 2014; 22:2018-2027; Hinderer, et al, Molecular therapy: thejournal of the American Society of Gene Therapy 2015; 23:1298-1307;Gurda, et al, Molecular therapy: the journal of the American Society ofGene Therapy 2015; Higuchi et al, cited above; Haurigot, et al, J ClinInvest, 2013: 123: 3254-3271; Gray et al, Gene Therapy, 2013: 20:450-459]. Notably, liver transduction after IT AAV vector deliveryvaries substantially among species, so it is not yet clear whetherhumans would exhibit significant peripheral expression.

This study demonstrates that IT AAV9 delivery achieves effective IDSgene transfer to the brain and resolve CNS manifestation of MPS II,supporting the advancement of this approach into the clinic.

These data provide preliminary evidence that AAV9.CB.hIDS can improveneurocognitive deficits in MPS II mice.

Example 3: Non-Human Primate Studies

A study in non-human primates (NHPs) is performed to evaluate the safetyand effect of AAV9.CB.hIDS in adult male rhesus macaques. The objectiveof the study is to evaluate local, acute, and chronic toxicities ofintrathecal (IT) AAV9.CB.hIDS and to define the biodistribution of thevector. A total of 21 male macaques are treated with an IT injection ofAAV9.CB.hIDS at one of two doses of AAV9.CB.hIDS (5.6×10¹¹ or 1.875×10¹¹GC/g brain mass) or with vehicle control diluent. The low dose isapproximately 100-fold greater than the MED in MPS II mice (MED of1.875×10⁹ GC/g) and corresponds to the lowest dose producingstatistically significant histological improvements in MPS II mice andneurobehavioral changes thought to reflect cognitive function. Thehigher dose (approximately 300× the MED) is the maximal concentration atwhich the vector can be formulated and is limited by the requirement tomaintain an injection volume that can be safely administered to the CSF(<10% CSF volume). During the injection procedure, animals aremaintained under general anesthesia. A spinal needle is placed into thesuboccipital space; placement is verified via fluoroscopy, and the doseis administered in a total volume of 1 mL. Serum and CSF is collectedfor clinical pathology assessments at Days 0, 3, 7, 14, 21 and 30 afterinjection and monthly thereafter. On Days 14, 90, and 180 afterinjection, 3 male macaques from each dose group (6 total per time point)are sacrificed for pathology and biodistribution. Vehicle treatedanimals serve as controls. Full histopathology is performed on tissuesfrom all animals, and analysis of vector biodistribution is conductedfor the high-dose cohort. DNA extraction and detection of vector genomesby quantitative PCR are performed as previously described (see Chen etal, 2013, Hum Gene Ther Clin Dev, 24(4): 154-160). This assay has asensitivity greater than 20 vector genome copies per μg of DNA, andspike controls are included to verify reaction efficiency in individualsamples. Biodistribution, biochemistry, and immunogenicity data iscollected at 14 days, 3 months and 6 months.

Example 4: Manufacture of rAAV9.CB7.hIDS Vector

The AAV9.CB7.hIDS is produced by triple plasmid transfection of humanHEK293 MCB cells with: (i) the hIDS vector genome plasmid, (ii) an AAVhelper plasmid termed pAAV29 containing the AAV rep2 and cap 9 wild-typegenes and (iii) a helper adenovirus plasmid termed pAdΔF6(Kan).

Cloning of the plasmid pAAV.CB7.CI.hIDS.RGB is as described in Example 1above. The vector genome derived from this plasmid is a single-strandedDNA genome with AAV2 derived ITRs flanking the hIDS expression cassette.Expression from the transgene cassette is driven by a CB7 promoter, ahybrid between a cytomegalovirus (CMV) immediate early enhancer (C4) andthe chicken beta actin promoter, while transcription from this promoteris enhanced by the presence of the chicken beta actin intron (CI). ThepolyA signal for the expression cassette is the rabbit beta-globin (RBG)polyA. The plasmid was constructed by codon-optimizing and synthesizingthe hIDS sequence [nt 1177 to nt 2829 of SEQ ID NO: 8, and nt 1937 to nt3589 of SEQ ID NO: 11] and the resulting construct was then cloned intothe plasmid pENN.AAV.CB7.CI.RBG (p1044), an AAV2 ITR-flanked expressioncassette containing CB7, CI and RBG expression elements to givepAAV.CB7.CI.hIDS.RBG.

Cloning of the cis plasmid pAAV.CB7CIhIDS.RGB.KanR: The vector genomewas excised from this plasmid using the Pad restriction enzyme andcloned into a pKSS-based plasmid backbone (p2017) containing thekanamycin resistance gene. The final vector genome plasmid ispAAV.CB7.CI.hIDS.RBG.KanR.

AAV2/9 helper plasmid pAAV29KanRGXRep2: The AAV2/9 helper plasmidpAAV29KanRGXRep2 encodes the 4 wild-type AAV2 rep proteins and the 3wild-type AAV VP capsid proteins from AAV9. To create the chimericpackaging construct, first the AAV2 cap gene from plasmid p5E18,containing the wild type AAV2 rep and cap genes, was removed andreplaced with a PCR fragment of the AAV9 cap gene amplified from liverDNA. The resulting plasmid was given the identifier pAAV2-9 (p0008).Note that the AAV p5 promoter which normally drives rep expression ismoved in this construct from the 5′ end of rep to the 3′ end of cap.This arrangement serves to introduce a spacer between the promoter andthe rep gene (i.e. the plasmid backbone), down-regulate expression ofrep and increase the ability to support vector production. The plasmidbackbone in p5E18 is from pBluescript KS. The AAV2/9 helper plasmidpAAV29KanRGXRep2 encodes the 4 wild-type AAV2 rep proteins, the 3wild-type AAV VP capsid proteins from AAV9, and kanamycin resistance.

pAdDeltaF6(Kan) adenovirus helper plasmid is 15,770 bp in size. Theplasmid contains the regions of adenovirus genome that are important forAAV replication, namely E2A, E4, and VA RNA (the adenovirus E1 functionsare provided by the 293 cells), but does not contain other adenovirusreplication or structural genes. The plasmid does not contain the ciselements critical for replication such as the adenoviral invertedterminal repeats and therefore, no infectious adenovirus is expected tobe generated. It was derived from an E1, E3 deleted molecular clone ofAd5 (pBHG10, a pBR322 based plasmid). Deletions were introduced in theAd5 DNA to remove expression of unnecessary adenovirus genes and reducethe amount of adenovirus DNA from 32 Kb to 12 kb. Finally the ampicillinresistance gene was replaced by the kanamycin resistance gene to givepAdΔF6 (Kan). The functional elements of the E2, E4 and VAI adenoviralgenes necessary for AAV vector production remain in this plasmid. Theadenoviral E1 essential gene functions are supplied by the HEK293 cells.DNA plasmid sequencing was performed by Qiagen Genomic Services andrevealed 100% homology with the following important functional elementsof the reference sequence pAdDeltaF6(Kan) p1707FH-Q: E4 ORF6 3692-2808bp; E2A DNA binding protein 11784-10194 bp; VA RNA region 12426-13378bp.

A flow diagram summarizing the manufacturing process is provided inFIGS. 10A-10B.

Cell Seeding: A qualified human embryonic kidney 293 cell line will beused for the production process. Cells will be expanded to 5×10⁹-5×10¹⁰cells using Corning T-flasks and CS-10, which will allow sufficient cellmass to be generated for seeding up to 50 HS-36 for vector productionper BDS lot. Cells will be cultivated in medium composed of Dulbecco'sModified Eagle Medium (DMEM), supplemented with 10% gamma irradiated,US-sourced, Fetal Bovine Serum (FBS). The cells are anchorage dependentand cell disassociation will be accomplished using TrypLE Select, ananimal product-free cell dissociation reagent. Cell seeding isaccomplished using sterile, single-use disposable bioprocess bags andtubing sets. The cells are maintained at 37° C. (±2° C.), in 5% (±0.5%)CO₂ atmosphere. Cell culture media is replaced with fresh, serum freeDMEM media and transfected with the three production plasmids using anoptimized PEI-based transfection method. All plasmids used in theproduction process are produced in the context of a CMO quality systemand infrastructure utilizing the most salient features of cGMPmanufacturing; traceability, document control, and materialssegregation.

Sufficient DNA plasmid transfection complex is prepared in the BSC totransfect up to 50 HS-36 (per BDS batch). Initially a DNA/PEI mixture isprepared containing 7.5 mg of pAAV.CB7.CI.hIDS.RBG.KanR vector genomeplasmid, 150 mg of pAdDeltaF6(Kan), 75 mg of pAAV29KanRGXRep2 AAV helperplasmid and GMP grade PEI (PEIPro, PolyPlus Transfection SA). Thisplasmid ratio was determined to be optimal for AAV production in smallscale optimization studies. After mixing well, the solution is allowedto sit at room temperature for 25 min and then added to serum-free mediato quench the reaction and then added to the HS-36's. The transfectionmixture is equalized between all 36 layers of the HS-36 and the cellsare incubated at 37° C. (±2° C.) in a 5% (±0.5%) CO₂ atmosphere for 5days.

Cell Media Harvesting: Transfected cells and media are harvested fromeach HS-36 using disposable bioprocess bags by aseptically draining themedium out of the units. Following the harvest of media, the ˜80 litervolume is supplemented with MgCl₂ to a final concentration of 2 mM(co-factor for Benzonase) and Benzonase nuclease (Cat #: 1.016797.0001,Merck Group) are added to a final concentration of 25 units/ml. Theproduct (in a disposable bioprocess bag) is incubated at 37° C. for 2 hrin an incubator to provide sufficient time for enzymatic digestion ofresidual cellular and plasmid DNA present in the harvest as a result ofthe transfection procedure. This step is performed to minimize theamount of residual DNA in the final vector. After the incubation period,NaCl is added to a final concentration of 500 mM to aid in the recoveryof the product during filtration and downstream tangential flowfiltration.

Clarification: Cells and cellular debris are removed from the productusing a depth filter capsule (1.2 μm/0.22 um) connected in series as asterile, closed tubing and bag set that is driven by a peristaltic pump.Clarification assures that downstream filters and chromatography columnsare protected from fouling and bioburden reduction filtration ensuresthat at the end of the filter train, any bioburden potentiallyintroduced during the upstream production process is removed beforedownstream purification. The harvest material is passed through aSartorius Sartoguard PES capsule filter (1.2/0.22 μm) (Sartorius StedimBiotech Inc.).

Large-scale Tangential Flow Filtration: Volume reduction (10-fold) ofthe clarified product is achieved by Tangential Flow Filtration (TFF)using a custom sterile, closed bioprocessing tubing, bag and membraneset. The principle of TFF is to flow a solution under pressure parallelto a membrane of suitable porosity (100 kDa). The pressure differentialdrives molecules of smaller size through the membrane and effectivelyinto the waste stream while retaining molecules larger than the membranepores. By recirculating the solution, the parallel flow sweeps themembrane surface preventing membrane pore fouling. By choosing anappropriate membrane pore size and surface area, a liquid sample may berapidly reduced in volume while retaining and concentrating the desiredmolecule. Diafiltration in TFF applications involves addition of a freshbuffer to the recirculating sample at the same rate that liquid ispassing through the membrane and to the waste stream. With increasingvolumes of diafiltration, increasing amounts of the small molecules areremoved from the recirculating sample. This results in a modestpurification of the clarified product, but also achieves buffer exchangecompatible with the subsequent affinity column chromatography step.Accordingly, we utilize a 100 kDa, PES membrane for concentration thatis then diafiltrated with 4 volumes of a buffer composed of: 20 mM TrispH 7.5 and 400 mM NaCl. The diafiltered product is stored overnight at4° C. and then further clarified with a 1.2 μm/0.22 um depth filtercapsule to remove any precipitated material.

Affinity Chromatography: The diafiltered product is applied to a CaptureSelect™ Poros-AAV2/9 affinity resin (Life Technologies) that efficientlycaptures the AAV2/9 serotype. Under these ionic conditions, asignificant percentage of residual cellular DNA and proteins flowthrough the column, while AAV particles are efficiently captured.Following application, the column is washed to remove additional feedimpurities followed by a low pH step elution (400 mM NaCl, 20 mM SodiumCitrate; pH 2.5) that is immediately neutralized by collection into a1/10th volume of a neutralization buffer (Bis Tris Propane, 200 mM, pH10.2).

Anion Exchange Chromatography: To achieve further reduction ofin-process impurities including empty AAV particles, the Poros-AAV2/9elution pool is diluted 50-fold (20 mM Bis Tris Propane, 0.001% PluronicF68; pH 10.2) to reduce ionic strength to enable binding to a CIMultus Qmonolith matrix (BIA Separations). Following a low-salt wash, vectorproduct is eluted using a 60 CV NaCl linear salt gradient (10-180 mMNaCl). This shallow salt gradient effectively separates capsid particleswithout a vector genome (empty particles) from particles containingvector genome (full particles) and results in a preparation enriched forfull capsids. Fractions are collected into tubes containing 1/100thvolume of 0.1% pluronic F68 and 1/27th volume of Bis Tris pH 6.3 tominimize non-specific binding to tubes and the length of exposure tohigh pH respectively. The appropriate peak fraction is collected, andthe peak area assessed and compared to previous data for determinationof the approximate vector yield.

Final Formulation and Sterile Filtration to yield the BDS: TFF is usedto achieve final formulation on the pooled AEX fractions with a 100 kDamembrane. This is achieved by diafiltration with 4 volumes offormulation buffer (Elliots B solution, 0.001% Pluronic F68) andconcentrated to yield the BDS, whereby the peak area from the anionexchange chromatography is compared to previous data in order toestimate the concentration factor to achieve a titer of ≥5×10¹³ GC/ml.Samples are removed for BDS testing (described in the section below).The filtered Purified Bulk is stored in sterile polypropylene tubes andfrozen at ≤−60° C. in a quarantine location until release for FinalFill. Preliminary stability study indicates that the DP does not loseactivity following freezing and thawing in our proposed formulationbuffer. Additional studies are underway to assess stability followingprolonged storage at −80° C.

Final Fill: The frozen BDS is thawed, pooled, diluted to the targettiter using the final formulation buffer, terminally filtered through a0.22 um filter (Millipore, Billerica, Mass.) and filled into WestPharmaceutical's “Ready-to-Use” (pre-sterilized) 2 ml glass vials and 13mm stoppers and seals at a fill volume ≥0.6 ml to ≤2.0 ml per vial.Individually labeled vials are labeled according to the specificationsbelow. Labeled vials are stored at ≤−60° C.

The vector (drug product) is vialed at a single fixed concentration andthe only variable is the volume per vial. To achieve lower doseconcentrations the drug product is diluted with Elliots B solution,0.001% Pluronic F68. The high dose vector is used directly withoutdilution while the low vector requires a 1:5 dilution in the formulationbuffer which is conducted by the pharmacy at the time of dosing.

Example 5: Testing of Vector

Characterization assays including serotype identity, empty particlecontent and transgene product identity are performed. Descriptions ofthe assays appear below.

A. Vector Genome Identity: DNA Sequencing

Viral Vector genomic DNA is isolated and the sequence determined by2-fold sequencing coverage using primer walking. Sequence alignment isperformed and compared to the expected sequence.

B. Vector Capsid Identity: AAV Capsid Mass spectrometry of VP3

Confirmation of the AAV2/9 serotype of the vector is achieved by anassay based upon analysis of peptides of the VP3 capsid protein by massspectrometry (MS). The method involves multi-enzyme digestion (trypsin,chymotrypsin and endoproteinase Glu-C) of the VP3 protein band excisedfrom SDS-PAGE gels followed by characterization on a UPLC-MS/MS on aQ-Exactive Orbitrap mass spectrometer to sequence the capsid protein. Atandem mass spectra (MS) method was developed that allows forsubtraction of the host protein products and deriving capsid peptidesequence from mass spectra.

C. Genomic Copy (GC) Titer

The oqPCR based genomic copy titer is determined over a range of serialdilutions and compared to the cognate plasmid standard(pAAV.CB7.CI.hIDS.RBG.KanR). The oqPCR assay utilizes sequentialdigestion with DNase I and Proteinase K, followed by qPCR analysis tomeasure encapsidated vector genomic copies. DNA detection isaccomplished using sequence specific primers targeting the RBG polyAregion in combination with a fluorescently tagged probe hybridizing tothis same region. Comparison to the plasmid DNA standard curve allowstiter determination without the need of any post-PCR samplemanipulation. A number of standards, validation samples and controls(for background and DNA contamination) have been introduced into theassay. The assay is qualified by establishing and defining assayparameters including sensitivity, limit of detection, range ofqualification and intra and inter assay precision. An internal AAV9reference lot is established and used to perform the qualificationstudies. Note that our previous experience suggests that the titerobtained by the optimized qPCR assay described here is generally 2.5fold higher than that achieved by our standard qPCR technique which wasused for the generation of the pre-clinical data.

D. Empty to Full Particle Ratio

The total particle content of the drug product is determined by SDS-PAGEanalysis. A reference vector preparation purified on an iodixanolgradient is analyzed by various methods (analytical ultracentrifugation,electron microscopy and absorbance at 260/280 nm) to established thatthe preparation contains >95% genome-containing (full) particles. Thisreference material is serially diluted to known genome copy numbers (andthus by extension, particle numbers) and each dilution is run on an SDSPAGE gel along with a similar dilution series of the drug product. Peakarea volumes of both the reference material and drug product VP3 proteinbands are determined by densitometry and the reference material volumesare plotted versus particle number. The total particle concentration ofthe drug product is determined by extrapolation from this curve and thegenome copy (GC) titer is then subtracted to obtain the empty particletiter. The empty to full particle ratio is the ratio of the emptyparticle titer to the GC titer.

E. Infectious Titer

The infectious unit (IU) assay is used to determine the productiveuptake and replication of vector in RC32 cells (rep2 expressing HeLacells). A 96-well end-point format has been employed similar to thatpreviously published. Briefly, RC32 cells are co-infected by serialdilutions of rAAV9.CB.hIDS and a uniform dilution of Ad5 with 12replicates at each dilution of rAAV. Seventy-two hours after infectionthe cells are lysed, and qPCR performed to detect rAAV vectoramplification over input. An end-point dilution TCID50 calculation(Spearman-Karber) is performed to determine a replicative titerexpressed as IU/ml. Since “infectivity” values are dependent onparticles coming into contact with cells, receptor binding,internalization, transport to the nucleus and genome replication, theyare influenced by assay geometry and the presence of appropriatereceptors and post-binding pathways in the cell line used. Receptors andpost-binding pathways are not usually maintained in immortalized celllines and thus infectivity assay titers are not an absolute measure ofthe number of “infectious” particles present. However, the ratio ofencapsidated GC to “infectious units” (described as GC/IU ratio) can beused as a measure of product consistency from lot to lot.

The GC/IU ratio is a measure of product consistency. The oqPCR titer(GC/ml) is divided by the infectious unit (IU/ml) to give the calculatedGC/IU ratio.

F. Replication-Competent AAV (rcAAV) Assay

A sample is analyzed for the presence of replication competent AAV2/9(rcAAV) that can potentially arise during the production process. A 3passage assay has been developed consisting of cell-based amplificationand passage followed by detection of rcAAV DNA by real-time qPCR (cap 9target). The cell-based component consists of inoculating monolayers ofHEK293 cells (P1) with dilutions of the test sample and wild-type humanadenovirus type 5 (Ad5). 10¹⁰ GC of the vector product is the maximalamount of the product tested. Due to the presence of adenovirus,replication competent AAV amplifies in the cell culture. After 2 days, acell lysate is generated and Ad5 heat inactivated. The clarified lysateis then passed onto a second round of cells (P2) to enhance sensitivity(again in the presence of Ad5). After 2 days, a cell lysate is generatedand Ad5 heat inactivated. The clarified lysate is then passed onto athird round of cells (P3) to maximize sensitivity (again in the presenceof Ad5). After 2 days, cells are lysed to release DNA which is thensubjected to qPCR to detect AAV9 cap sequences. Amplification of AAV9cap sequences in an Ad5 dependent manner indicates the presence ofrcAAV. The use of a AAV2/9 surrogate positive control containing AAV2rep and AAV9 cap genes enables the Limit of Detection (LOD) of the assayto be determined (0.1, 1, 10 and 100 IU) and using a serial dilution ofrAAV9.CB.hIDS vector (1×10¹⁰, 1×10⁹, 1×10⁸, 1×10⁷ GC) the approximatelevel of rcAAV present in the test sample can be quantitated.

G. In Vitro Potency

To relate the qPCR GC titer to gene expression, an in vitro bioassay isperformed by transducing HEK293 (Human Embryonic Kidney) cells with aknown multiplicity of GCs per cell and assaying the supernatant for IDSactivity 72 hours post-transduction. IDS activity is measured byincubating sample diluted in 0.1 ml water with 0.1 ml of 100 mmol/l4MU-iduronide-2-sulfate at 37 degrees for 1-3 hours. The reaction isstopped by the addition of 2 ml 290 mmol/l glycine, 180 mmol/l sodiumcitrate, pH 10.9 and liberated 4MU is quantified by comparingfluorescence to standard dilutions of 4MU.

Comparison to highly active pre-clinical and to x vector preparationsenables interpretation of product activity.

H. Total Protein, Capsid protein, Protein Purity Determination andCapsid Protein Ratio

Vector samples are first quantified for total protein against a BovineSerum Albumin (BSA) protein standard curve using a bicinchoninic acid(BCA) assay. The determination is made by mixing equal parts of samplewith a Micro-BCA reagent provided in the kit. The same procedure isapplied to dilutions of a BSA Standard. The mixtures are incubated at60° C. and absorbance measured at 562 nm. A standard curve is generatedfrom the standard absorbance of the known concentrations using a4-Parameter fit. Unknown samples are quantified according to the4-Parameter regression.

To provide a semi-quantitative determination of AAV purity, the samplesare then normalized for genome titer and 5×10⁹ GC separated on anSDS-polyacrylamide (SDS-PAGE) gel under reducing conditions. The gel isthen stained with SYPRO Ruby dye. Any impurity bands are quantified bydensitometry by comparison to co-electrophoresed BSA standards of 25,50, and 100 ng of protein per lane. These quantities represent 1%, 2%and 4% of the total AAV protein sample. Stained bands that appear inaddition to the three AAV specific proteins VP1, VP2 and VP3 areconsidered protein impurities. All impurity bands are compared to thereference proteins and the impurity mass percent as well as approximatemolecular weight are reported. The SDS-PAGE gels are also used toquantify the VP1, VP2 and VP3 proteins and determine their ratio.

Example 6: MPS II Biomarker

In the present study, metabolite profiling of CSF samples from MPS Idogs was performed, which revealed substantial disease relatedalterations in the CSF metabolome. The most striking difference was anover 30-fold elevation in spermine levels compared to normal controls.This finding was confirmed in MPS I patient samples, as well as in afeline model of MPS I and is expected to be found also in MPS II.Spermine binds to HS, and cellular uptake of spermine is dependent onthis interaction [M. Belting, S. Persson, L.-Å. Fransson, Proteoglycaninvolvement in polyamine uptake. Biochemical Journal 338, 317-323(1999); J. E. Welch, P. Bengtson, K. Svensson, A. Wittrup, G. J.Jenniskens, G. B. Ten Dam, T. H. Van Kuppevelt, M. Belting, Single chainfragment anti-heparan sulfate antibody targets the polyamine transportsystem and attenuates polyamine-dependent cell proliferation.International journal of oncology 32, 749-756 (2008); published onlineEpubApr]. Cell surface proteoglycans such as glypican-1 can bindspermine through their HS moieties, and after endocytosis of theglypican protein, intracellular cleavage of the HS chain releases boundspermine into the cell (Belting et al; K. Ding, S et al, The Journal ofbiological chemistry 276, 46779-46791 (2001); published online EpubDec14). Thus, intact HS recycling is essential for spermine uptake. In MPSI, extracellular spermine accumulation could occur through inhibition ofthis uptake mechanism due to inefficient HS recycling, or through simplebinding of spermine to the extracellular GAGs that accumulate in MPS,shifting the spermine binding equilibrium to favor extracellulardistribution. Future studies should address the relative contribution ofthese mechanisms to spermine accumulation in MPS I CSF.

We found that inhibitors of spermine synthesis blocked excess neuritegrowth in MPS neurons, and that neurite growth could be induced in WTneurons by spermine concentrations similar to those found in patientCSF. Gene therapy in the dog model of MPS I reversed spermineaccumulation and normalized expression of GAP43, suggesting that thesame pathway was impacted in vivo. We could not directly evaluate theimpact of spermine synthesis inhibition in vivo, as available inhibitorsdo not cross the blood-brain barrier, and chronic direct CNSadministration from birth is not feasible in our animal models. Whileour in vitro findings support a role for spermine in aberrant neuritegrowth in MPS I, it is important to note that inhibiting sperminesynthesis did not completely reverse the phenotype, and spermineaddition to normal neurons did not increase neurite growth to the levelof MPS I neurons. The effects of spermine modulation may have beenlimited by the relatively short period of treatment. It is also possiblethat spermine accumulation is not the sole mediator contributing toneurite outgrowth in MPS I. Notably many neurotrophic factors bindthrough HS modified receptors, and interactions with HS in extracellularmatrix can influence neurite growth [D. Van Vactor, D. P. Wall, K. G.Johnson, Heparan sulfate proteoglycans and the emergence of neuronalconnectivity. Current opinion in neurobiology 16, 40-51 (2006);published online EpubFeb (10.1016/j.conb.2006.01.011)]. Spermineaccumulation may therefore be one of several factors promoting abnormalneurite growth in MPS I.

Of the 15 MPS I dog CSF samples screened, only one fell within thenormal range of spermine concentration. At 28 days of age, this was theyoungest animal included in the study. This finding indicates thatspermine accumulation may be age dependent. Future studies shouldevaluate CSF spermine levels longitudinally in MPS patients. If spermineincreases with age in MPS patients, this could explain the kinetics ofcognitive decline, as most patients experience 1-2 years of normaldevelopment before the onset of developmental delays.

The potential for impaired HS metabolism to trigger accumulation of ametabolite that alters neuron growth could point to a novel connectionbetween enzyme deficiencies and the abnormal neurite growth phenotype inMPS I, which may explain the cognitive dysfunction associated with thesedisorders. These findings also indicate that CSF spermine is useful as anoninvasive biomarker for assessing pharmacodynamics of novelCNS-directed therapies for MPSI.

Materials and Methods:

Experimental design: This study was initially designed to detectmetabolites that were present at significantly different levels in MPS Ipatient CSF samples compared to samples from healthy controls. Due tothe limited availability of CSF samples from children with MPS IH andhealthy controls, the initial screen was performed using CSF samplesfrom MPS I dogs, for which greater numbers were available, with theintention of subsequently evaluating candidate biomarkers in humansamples. A total of 15 CSF samples from individual untreated MPS I dogswere available for analysis, and an additional 15 samples were obtainedfrom healthy controls. Following identification of elevated spermine inMPS I dog CSF in the prospective metabolite screen, spermine wasretrospectively measured in CSF samples from previous studies of MPS Idogs and cats treated with gene therapy, as well as patient samples. Thenumber of subjects included in each group for these analyses was limitedby sample availability and was not based on statistical considerations;therefore in some cases numbers were insufficient for statisticalcomparisons. For studies of in vitro neurite growth, the number of cellsquantified for each condition was based on pilot experiments whichindicated that >30 cells per condition was required to detect a 20%difference in arbor length, neurite number or neurite branches per cell.After cells were plated and treated with the designated drug, the wellswere coded and the acquisition of cell images and the manualquantification of neurite length and branching were performed by ablinded reviewer. The comparison of wildtype and MPS mouse neurons wasrepeated using a different substrate [poly-L-lysine (Sigma) coatedtissue culture plates rather than chamber slides (Sigma S6815)] withsimilar results. The comparison of wildtype neurons with and withoutspermine addition was performed four times using both substrates withsimilar results.

CSF metabolite profiling: CSF metabolite profiling was performed byMetabolon.

Samples were stored at −80° C. until processing. Samples were preparedusing the MicroLab STAR® system (Hamilton Company). A recovery standardwas added prior to the first step in the extraction process for QCpurposes. Proteins were precipitated with methanol under vigorousshaking for 2 min followed by centrifugation. The resulting extract wasdivided into five fractions: one for analysis by reverse phase (RP)UPLC-MS/MS with positive ion mode electrospray ionization, one foranalysis by RP/UPLC-MS/MS with negative ion mode electrosprayionization, one for analysis by hydrophilic interaction chromatography(HILIC)/UPLC-MS/MS with negative ion mode electrospray ionization, onefor analysis by GC-MS, and one sample was reserved for backup. Sampleswere placed briefly on a TurboVap® (Zymark) to remove the organicsolvent. For LC, the samples were stored overnight under nitrogen beforepreparation for analysis. For GC, each sample was dried under vacuumovernight before preparation for analysis.

The LC/MS portion of the platform was based on a Waters ACQUITYultra-performance liquid chromatography (UPLC) and a Thermo ScientificQ-Exactive high resolution/accurate mass spectrometer interfaced with aheated electrospray ionization (HESI-II) source and Orbitrap massanalyzer operated at 35,000 mass resolution. The sample extract wasdried then reconstituted in solvents compatible to each of the LC/MSmethods. Each reconstitution solvent contained a series of standards atfixed concentrations to ensure injection and chromatographic consistencyFor RP chromatography, one aliquot was analyzed using acidic positiveion optimized conditions and the other using basic negative ionoptimized conditions Each method utilized separate dedicated columns(Waters UPLC BEH C18-2.1×100 mm, 1.7 μm). The extracts reconstituted inacidic conditions were gradient eluted using water and methanolcontaining 0.1% formic acid. The basic extracts were similarly elutedusing methanol and water, however with 6.5 mM ammonium bicarbonate. Thethird aliquot was analyzed via negative ionization following elutionfrom a HILIC column (Waters UPLC BEH Amide 2.1×150 mm, 1.7 μm) using agradient consisting of water and acetonitrile with 10 mM ammoniumformate. The MS analysis alternated between MS and data-dependent MSnscans using dynamic exclusion. The scan range varied slightly betweenmethods but covered 80-1000 m/z.

The samples destined for analysis by GC-MS were dried under vacuum for aminimum of 18 h prior to being derivatized under dried nitrogen usingbistrimethyl-silyltrifluoroacetamide. Derivatized samples were separatedon a 5% diphenyl/95% dimethyl polysiloxane fused silica column (20m×0.18 mm ID; 0.18 um film thickness) with helium as carrier gas and atemperature ramp from 60° to 340° C. in a 17.5 min period. Samples wereanalyzed on a Thermo-Finnigan Trace DSQ fast-scanning single-quadrupolemass spectrometer using electron impact ionization (EI) and operated atunit mass resolving power. The scan range was from 50-750 m/z.

Several types of controls were analyzed in concert with the experimentalsamples: a pooled matrix sample generated by taking a small volume ofeach experimental sample served as a technical replicate throughout thedata set; extracted water samples served as process blanks; and acocktail of QC standards that were carefully chosen not to interferewith the measurement of endogenous compounds were spiked into everyanalyzed sample, allowed instrument performance monitoring and aidedchromatographic alignment. Instrument variability was determined bycalculating the median relative standard deviation (RSD) for thestandards that were added to each sample prior to injection into themass spectrometers. Overall process variability was determined bycalculating the median RSD for all endogenous metabolites (i.e.,non-instrument standards) present in 100% of the pooled matrix samples.Experimental samples were randomized across the platform run with QCsamples spaced evenly among the injections.

Metabolites were identified by automated comparison of the ion featuresin the experimental samples to a reference library of chemical standardentries that included retention time, molecular weight (m/z), preferredadducts, and in-source fragments as well as associated MS spectra andcurated by visual inspection for quality control using softwaredeveloped at Metabolon. Identification of known chemical entities wasbased on comparison to metabolomics library entries of purifiedstandards. Peaks were quantified using area-under-the-curvemeasurements. Raw area counts for each metabolite in each sample werenormalized to correct for variation resulting from instrument inter-daytuning differences by the median value for each run-day, therefore,setting the medians to 1.0 for each run. This preserved variationbetween samples but allowed metabolites of widely different raw peakareas to be compared on a similar graphical scale. Missing values wereimputed with the observed minimum after normalization.

Quantitative MS assay: CSF samples (50 μL) were mixed with a spermine-d8internal standard (IsoSciences). Samples were deproteinized by mixingwith a 4-fold excess of methanol and centrifuging at 12,000×g at 4° C.The supernatant was dried under a stream of nitrogen, and thenresuspended in 50 μL of water. An aliquot of 5 μL was subjected to LC-MSanalysis. The LC separations were carried out using a Waters ACQUITYUPLC system (Waters Corp., Milford, Mass., USA) equipped with anXbridge® C18 column (3.5 μm, 150×2.1 mm). The flow-rate was 0.15 mL/min,solvent A was 0.1% formic acid and solvent B was 98/2 acetonitrile/H₂O(v/v) with 0.1% formic acid. The elution conditions were as follows: 2%B at 0 min, 2% B at 2 min, 60% B at 5 min, 80% B at 10 min, 98% B at 11min, 98% B at 16 min, 2% B at 17 min, 2% B at 22 min, with the columntemperature being 35° C. A Finnigan TSQ Quantum Ultra spectrometer(Thermo Fisher, San Jose, Calif.) was used to conduct MS/MS analysis inpositive ion mode with the following parameters: spray voltage at 4000V, capillary temperature at 270° C., sheath gas pressure at 35 arbitraryunits, ion sweep gas pressure at 2 arbitrary units, auxiliary gaspressure at 10 arbitrary units, vaporizer temperature at 200° C., tubelens offset at 50, capillary offset at 35 and skimmer offset at 0. Thefollowing transitions were monitored: 203.1/112.1 (spermine);211.1/120.1 (spermine-d8) with scan width of 0.002 m/z, and scan timebeing 0.15 s.

Animal procedures: All animal protocols were approved by theInstitutional Animal Care and Use Committee of the University ofPennsylvania. For CSF metabolite screening, samples were collected bysuboccipital puncture in normal dogs at 3-26 months of age, and in MPS Idogs at 1-18 months of age. Gene transfer studies in MPS I dogs and catswere performed as previously described (20, 22). CSF samples werecollected 6-8 months after vector administration. For mouse corticalneuron experiments, primary cortical neuron cultures were prepared fromE18 IDUA−/− or IDUA+/+ embryos.

Patient samples: Informed consent was obtained from each subject'sparent or legal guardian. The protocol was approved by the InstitutionalReview Board of the University of Minnesota. CSF was collected by lumbarpuncture. All MPS I patients had a diagnosis of Hurler syndrome and hadnot received enzyme replacement therapy or hematopoietic stem celltransplantation prior to sample collection. MPS I patients were 6-26months of age. The healthy controls were 36 and 48 months of age.

Statistical analysis: The random forest analysis and heat map generationwere performed using MetaboAnalyst 3.0 [R. G. Kalb, Development 120,3063-3071 (1994); J. Zhong, et al, Journal of neurochemistry 64, 531-539(1995) D. Van Vactor, D. P. W et al, Current opinion in neurobiology 16,40-51 (2006); published online EpubFeb (10.1016/j.conb.2006.01.011). Rawpeak data were log transformed and normalized to the mean of normalsample values. All other statistical analyses were performed withGraphPad Prism 6. Cultured neuron arbor length, neurite number, andbranching were compared by ANOVA followed by Dunnett's test. CSFspermine and cortical GAP43 were compared by Kruskal-Wallis testfollowed by Dunn's test.

GAP43 western: Samples of frontal cortex were homogenized in 0.2% tritonX-100 using a Qiagen Tissuelyser at 30 Hz for 5 min Samples wereclarified by centrifugation at 4° C. Protein concentration wasdetermined in supernatants by BCA assay. Samples were incubated inNuPAGE LDS buffer with DTT (Thermo Fisher Scientific) at 70° C. for 1 hrand separated on a Bis-Tris 4-12% polyacrylamide gel in MOPS buffer.Protein was transferred to a PVDF membrane, and blocked for 1 hr in 5%nonfat dry milk. The membrane was probed with rabbit polyclonalanti-GAP43 antibody (Abcam) diluted to 1 μg/mL in 5% nonfat dry milkfollowed by an HRP conjugated polyclonal anti-rabbit antibody (ThermoFisher Scientific) diluted 1:10,000 in 5% nonfat dry milk. Bands weredetected using SuperSignal West Pico substrate (Thermo FisherScientific). Densitometry was performed using Image Lab 5.1 (Bio-Rad).

Neurite growth assay: Day 18 embryonic cortical neurons were harvestedas described above, and plated at a concentration of 100,000 cells/mL onchamber slides (Sigma S6815) or poly-L-lysine (Sigma) coated tissueculture plates in serum-free Neurobasal medium (Gibco) supplemented byB27 (Gibco). Treatments were applied to duplicate wells 24 hours afterplating (day 1). Phase-contrast images for quantification were taken ona Nikon Eclipse Ti at 20× using a 600 ms manual exposure and 1.70× gainon high contrast. An individual blind to treatment conditions captured10-20 images per well and coded them. Images were converted to 8-bitformat in ImageJ (NIH) and traced in NeuronJ [E. Meijering, M. Jacob, J.C. Sarria, P. Steiner, H. Hirling, M. Unser, Design and validation of atool for neurite tracing and analysis in fluorescence microscopy images.Cytometry. Part A: the journal of the International Society forAnalytical Cytology 58, 167-176 (2004); published online EpubApr(10.1002/cyto.a.20022)] by a blinded reviewer. Soma diameter, neuritenumber, branch points, and arbor length were traced manually. Imagestraced in NeuronJ were converted to micrometers using a conversionfactor based on image size; 2560×1920 pixel images were converted tomicrometers using a conversion factor of 0.17 micrometers/pixel.

Histology: Brain tissue processing and LIMP2 immunofluorescence wereperformed as previously described [C. Hinderer, et al, Moleculartherapy: the journal of the American Society of Gene Therapy 22,2018-2027 (2014); published online EpubDec (10.1038/mt.2014.135)].

RT-PCR: Samples of frontal cortex from 3 normal dogs and 5 MPS dogs wereimmediately frozen on dry ice at necropsy. RNA was extracted with TRIzolreagent (Thermo Fisher Scientific), treated with DNAse I (Roche) for 20min at room temperature, and purified using an RNeasy kit (Qiagen)according to the manufacturer's instructions. Purified RNA (500 ng) wasreverse transcribed using the High Capacity cDNA Synthesis Kit (AppliedBiosystems) with random hexamer primers. Transcripts for arginase,ornithine decarboxylase, spermine synthase, spermidine synthase,spermine-spermidine acetyltransferase and glyceraldehyde phosphatedehydrogenase were quantified by Sybr green PCR using an AppliedBiosystems 7500

Real-Time PCR System. A standard curve was generated for each targetgene using four-fold dilutions of a pooled standard comprised of allindividual samples. The highest standard was assigned an arbitrarytranscript number, and Ct values for individual samples were convertedto transcript numbers based on the standard curve. Values are expressedrelative to the GAPDH control.

Statistical analysis: Random forest analysis and heat map generationwere performed using MetaboAnalyst 3.0 [J. Xia, et al, MetaboAnalyst2.0—a comprehensive server for metabolomic data analysis. Nucleic AcidsResearch, (2012); published online EpubMay 2, 2012 (10.1093/nar/gks374);J. Xia, et al., MetaboAnalyst: a web server for metabolomic dataanalysis and interpretation. Nucleic Acids Research 37, W652-W660(2009); published online EpubJul. 1, 2009 (10.1093/nar/gkp356). J. Xia,et al, MetaboAnalyst 3.0—making metabolomics more meaningful. NucleicAcids Research, (2015); published online EpubApr. 20, 2015(10.1093/nar/gkv380)]. Undetectable values in the metabolite screen wereimputed with the minimum values observed in the data set. Raw peak datawere normalized to the mean of normal sample values and log transformed.All other statistical analyses were performed with GraphPad Prism 6.Cultured neuron arbor length, neurite number, and branching werecompared by ANOVA followed by Dunnett's test. CSF spermine and corticalGAP43 were compared by Kruskal-Wallis test followed by Dunn's test.

Results

1. Identification of Elevated CSF Spermine Through Metabolite Profiling

An initial screen of CSF metabolites was carried out using a caninemodel of MPS I. These animals carry a splice site mutation in the IDUAgene, resulting in complete loss of enzyme expression and development ofclinical and histological features analogous to those of MPS I patients[K. P. Menon, et al, Genomics 14, 763-768 (1992); R. Shull, et al., TheAmerican journal of pathology 114, 487 (1984)]. CSF samples werecollected from 15 normal dogs and 15 MPS I dogs. CSF samples wereevaluated for relative quantities of metabolites by LC and GC-MS. Atotal of 281 metabolites could be positively identified in CSF samplesby mass spectrometry. Of these, 47 (17%) were significantly elevated inMPS I dogs relative to controls, and 88 (31%) were decreased relative tocontrols. A heat map of the 50 metabolites most different between groupsis shown in FIG. 19A. Metabolite profiling identified marked differencesin polyamine, sphingolipid, acetylated amino acid, and nucleotidemetabolism between MPS I and normal dogs. Random forest clusteringanalysis identified the polyamine spermine as the largest contributor tothe metabolite differences between MPS I and normal dogs (FIG. 23). Onaverage spermine was more than 30-fold elevated in MPS I dogs, with theexception of one MPS I dog that was under 1 month of age at the time ofsample collection. A stable isotope dilution (SID)-LC-MS/MS assay wasdeveloped to quantitatively measure spermine in CSF. Samples werescreened from 6 children with Hurler syndrome (ages 6-26 months), aswell as 2 healthy controls (ages 36 and 48 months). Both healthycontrols had CSF spermine levels below the limit of quantification (1ng/mL) of the assay, whereas CSF samples from MPS I patients were onaverage 10-fold above the limit of quantification (FIG. 19B). Spermineelevation in MPS IH patients appeared consistent with the known role ofHS in spermine binding and uptake [M. Belting, et al, Journal ofBiological Chemistry 278, 47181-47189 (2003); M. Belting, et al,Proteoglycan involvement in polyamine uptake. Biochemical Journal 338,317-323 (1999); J. E. Welch, et al, International journal of oncology32, 749-756 (2008))]; increased synthesis appeared unlikely as a causeof elevated CSF spermine, as normal and MPS I dog brain samples hadsimilar mRNA expression levels for transcriptionally regulated enzymesin the polyamine synthetic pathway (FIG. 24). To determine whetherspermine elevation was a general property of heparan sulfate storagediseases or specific to MPS I, spermine was measured in a CSF samplefrom a canine model of MPS VII, which exhibited a similar elevation(FIG. 25).

2. Role of Spermine in Abnormal Neurite Growth Associated with MPS

Following axon injury neurons upregulate polyamine synthesis, whichpromotes neurite outgrowth [D. Cai, et al, Neuron 35, 711-719 (2002);published online EpubAug 15; K. Deng, et al, The Journal ofneuroscience: the official journal of the Society for Neuroscience 29,9545-9552 (2009); published online EpubJul 29; Y. Gao, et al, Neuron 44,609-621 (2004); published online EpubNov 18; R. C. Schreiber, et al.,Neuroscience 128, 741-749 (2004)]. We therefore evaluated the role ofspermine in the abnormal neurite overgrowth phenotype that has beendescribed in MPS neurons [Hocquemiller, S., et al, Journal ofneuroscience research 88, 202-213 (2010)]. Cultures of E18 corticalneurons from MPS I mice exhibited greater neurite number, branching, andtotal arbor length after 4 days in culture than neurons derived fromwild type mice from the colony (FIGS. 20A-20F). Treatment of MPS neuronswith APCHA, an inhibitor of spermine synthesis, significantly reducedneurite growth and branching. The effect was reversible by replacingspermine (FIGS. 20A-20F). The same APCHA concentration did not affectthe growth of normal neurons (FIG. 26). Addition of spermine to wildtype neuron cultures at concentrations similar to those identified invivo resulted in significant increases in neurite growth and branching(FIGS. 20A-20F).

3. Impact of Gene Therapy on CSF Spermine and GAP43 Expression

GAP43, a central regulator of neurite growth, is overexpressed by MPSIII mouse neurons both in vitro and in vivo, suggesting that the sameneurite growth pathway aberrantly activated in neuron cultures is alsoactive in vivo. In order to evaluate the effect of IDUA deficiency onGAP43 expression and spermine accumulation in vivo, we measured CSFspermine and brain GAP43 levels in untreated MPS I dogs as well as thosetreated with CNS directed gene therapy. We previously described five MPSI dogs that were treated with an intrathecal injection of anadeno-associated virus serotype 9 vector carrying the canine IDUAtransgene [C. Hinderer, et al, Molecular therapy: the journal of theAmerican Society of Gene Therapy 23, 1298-1307 (2015); published onlineEpub August]. MPS I dogs can develop antibodies to the normal IDUAenzyme, so two of the dogs were pre-treated as newborns with hepaticIDUA gene transfer to induce immunological tolerance to the protein.Both tolerized dogs exhibited brain IDUA activity well above normalfollowing AAV9 treatment. The three non-tolerized dogs exhibited varyinglevels of expression, with one animal reaching levels greater thannormal and the other two exhibiting expression near normal (FIG. 21A).CSF spermine reduction was inversely proportional to brain IDUAactivity, with a 3-fold reduction relative to untreated animals in thetwo dogs with the lowest IDUA expression, and more than 20-foldreduction in the animal with the highest expression (FIGS. 21A, 21B to21H and 21K). GAP43 was upregulated in frontal cortex of MPS I dogs, andexpression was normalized in all vector treated animals (FIGS. 21I-21J).

We further evaluated the relationship between CSF spermine levels andIDUA reconstitution in MPS I dogs treated with a range of vector doses.MPS I dogs previously tolerized to human IDUA by neonatal hepatic genetransfer were treated with intrathecal injection of an AAV9 vectorexpressing human IDUA at one of 3 doses (10¹⁰, 10¹¹, 10¹² GC/kg, n=2 perdose) [(C. Hinderer, et al, Neonatal tolerance induction enablesaccurate evaluation of gene therapy for MPS I in a canine model.Molecular Genetics and Metabolism,dx.doi.org/10.1016/j.ymgme.2016.06.006]. CSF spermine was evaluated 6months after injection (FIG. 22A). Reduction of CSF spermine was dosedependent, with animals at the mid and high vector doses reaching thenormal range, whereas CSF spermine was only partially reduced in the lowdose animals. For independent verification of the connection betweenIDUA deficiency and CSF spermine accumulation, we evaluated CSF sperminelevels in a feline model of MPS I. Using CSF samples from our previouslyreported gene therapy studies, we found that untreated MPS I catsexhibited elevated CSF spermine (FIG. 22B) [C. Hinderer, P. Bell, B. L.Gurda, Q. Wang, J. P. Louboutin, Y. Zhu, J. Bagel, P. O'Donnell, T.Sikora, T. Ruane, P. Wang, M. E. Haskins, J. M. Wilson, Intrathecal genetherapy corrects CNS pathology in a feline model ofmucopolysaccharidosis I. Molecular therapy: the journal of the AmericanSociety of Gene Therapy 22, 2018-2027 (2014); published online EpubDec(10.1038/mt.2014.135)]. Intrathecal administration of a high dose of anAAV9 vector expressing feline IDUA normalized CSF spermine levels (FIG.22B).

C. Discussion

In the present study we performed metabolite profiling of CSF samplesfrom MPS I dogs, which revealed substantial disease related alterationsin the CSF metabolome. The most striking difference was an over 30-foldelevation in spermine levels compared to normal controls. This findingwas confirmed in MPS I patient samples, as well as in a feline model ofMPS I and a canine model of MPS VII. Spermine binds directly to HS withhigh affinity, and cellular uptake of spermine is dependent on thisinteraction [M. Belting, S. PERSSON, L.-A. Fransson, Proteoglycaninvolvement in polyamine uptake. Biochemical Journal 338, 317-323(1999); J. E. Welch, et al, International journal of oncology 32,749-756 (2008)]. Cell surface proteoglycans such as glypican-1 can bindspermine through their HS moieties, and after endocytosis of theglypican protein, intracellular cleavage of the HS chain releases boundspermine into the cell [Belting et al, cited above; K. Ding, et al, TheJournal of biological chemistry 276, 46779-46791 (2001); publishedonline EpubDec 14]. Thus, intact HS recycling is essential for spermineuptake. Inefficient HS recycling due to IDUA deficiency could inhibitthis spermine uptake mechanism, leading to extracellular spermineaccumulation. Alternatively, extracellular GAGs may sequester spermine,shifting the equilibrium to favor extracellular distribution. Themethanol deproteinization step employed for LC-MS sample preparation inthis study also precipitates soluble HS, suggesting that the sperminedetected in CSF is unbound, and therefore that uptake inhibition ratherthan GAG binding is responsible for extracellular spermine accumulation[N. Volpi, Journal of chromatography. B, Biomedical applications 685,27-34 (1996); published online EpubOct 11]. Formation and maintenance offunctional neural networks requires precise control of neurite growthand synapse formation. During development, the CNS environment becomesincreasingly inhibitory to neurite formation, with myelin associatedproteins largely blocking neurite growth in the adult brain. Thisdevelopmental shift toward decreased neurite growth is paralleled by adecrease in GAP43 expression [S. M. De la Monte, et al, DevelopmentalBrain Research 46, 161-168 (1989); published online Epub4/1/]. Thepersistent GAP43 expression and exaggerated neurite outgrowth exhibitedby MPS neurons may interfere with this normal balance of inhibitory andgrowth promoting signals, resulting in abnormal connectivity andimpaired cognition. How HS storage leads to this increase in neuritegrowth has not been established. A number of studies have implicatedpolyamines in neurite outgrowth; following axon injury, therate-limiting enzymes for the synthesis of spermine and its precursorsputrescine and spermidine are elevated, allowing for enhanced neuriteoutgrowth even in the presence of inhibitory signals from myelin [Cia(2002), Deng (2009), Gao (2004), all cited above. Further, treatment ofneurons with putrescine induces neurite growth when injected directlyinto CSF, an effect that is blocked by inhibitors of spermine synthesis(Deng (2009) cited above). The mechanism by which polyamines exert theireffect on neurite growth is not known. One potential target is the NMDAreceptor, activation of which is potentiated by spermine binding (J.Lerma, Neuron 8, 343-352 (1992); published online Epub2//(http://dx.doi.org/10.1016/0896-6273(92)90300-3)). NMDA signalinginduces neurite outgrowth, and the spermine sensitive subunit of thereceptor is highly expressed during development [D. Georgiev, et al,Experimental cell research 314, 2603-2617 (2008); published onlineEpubAug 15 (10.1016/j.yexcr.2008.06.009); R. G. Kalb, Regulation ofmotor neuron dendrite growth by NMDA receptor activation. Development120, 3063-3071 (1994); J. Zhong, et al, Journal of neurochemistry 64,531-539 (1995). Notably many neurotrophic factors bind through HSmodified receptors, and interactions with HS in extracellular matrix caninfluence neurite growth (D. Van Vactor, et al, Current opinion inneurobiology 16, 40-51 (2006); published online EpubFeb]. Spermineaccumulation may therefore be one of several factors promoting abnormalneurite growth in MPS I. Of the 15 MPS I dog CSF samples screened, onlyone fell within the normal range of spermine concentration. At 28 daysof age, this was the youngest animal included in the study. This findingindicates that spermine accumulation may be age dependent, although thisstudy demonstrates that it is already elevated by 6 months of age ininfants with Hurler syndrome. Future studies evaluates CSF sperminelevels longitudinally in MPS patients. If spermine increases with age inMPS patients, this explains the kinetics of cognitive decline, as mostpatients experience 1-2 years of normal development before the onset ofdevelopmental delays. The potential for impaired HS metabolism totrigger accumulation of a metabolite that alters neuron growth points toa novel connection between enzyme deficiencies and the abnormal neuritegrowth phenotype in MPS, which may explain the cognitive dysfunctionassociated with these disorders. Future studies confirms spermineelevation in other MPSs, such as MPS II. These findings also indicatethat CSF spermine is useful as a noninvasive biomarker for assessingpharmacodynamics of novel CNS-directed therapies for MPS. Future trialsfor CNS directed therapies evaluates the correlation between cognitiveendpoints and changes in CSF spermine.

Example 7: CT Guided ICV Delivery Device

A. Pre-Procedural Screening Assessments

1. Protocol Visit 1: Screening

The principal investigator describes the screening process that leads upto the intracisternal (IC) procedure, the administration procedureitself, and all potential safety risks in order for the subject (ordesignated caregiver) to be fully informed upon signing the informedconsent.

The following is performed and provided to theneuroradiologist/neurosurgeon/anesthesiologist in their screeningassessment of subject eligibility for the IC procedure: Medical history;concomitant medications; physical exam; vital signs; electrocardiogram(ECG); and laboratory testing results.

2. Interval: Screening to Study Visit 2

In order to allow adequate time to review eligibility, the followingprocedures is performed at any time between the first screening visitand up to one week prior to study Visit 2 (Day 0):

Head/Neck Magnetic Resonance Imaging (MRI) with and without gadolinium[note: Subject must be suitable candidate to receive gadolinium (i.e.,eGFR>30 mL/min/1.73 m²)]

In addition to the Head/Neck MRI, the investigator determines the needfor any further evaluation of the neck via flexion/extension studies

MRI protocol includes T1, T2, DTI, FLAIR, and CINE protocol images

Head/Neck MRA/MRV as per institutional protocol (note: Subjects with ahistory of intra/transdural operations may be excluded or may needfurther testing (e.g., radionucleotide cisternography) that allows foradequate evaluation of CSF flow and identification of possible blockageor lack of communication between CSF spaces.

Neuroradiologist/neurosurgeon subject procedural evaluation meeting: Therepresentatives from the 3 sites have a conference call (or web-meeting)to discuss the eligibility of each subject for the IC procedures basedon all available information (scans, medical history, physical exam,labs, etc.). All attempts should be made to achieve consensus onproceeding forward with the IC procedure or screen failing the subject(i.e., each member should be prepared to accept the decision made).

Anesthesia pre-op evaluation Day −28 to Day 1, with detailed assessmentof airway, neck (shortened/thickened) and head range-of-motion (degreeof neck flexion), keeping in mind the special physiologic needs of theMPS subject.

3. Day 1: Computerized Tomography Suite & Vector Preparation forAdministration. Prior to the IC procedure, the CT Suite confirms thefollowing equipment and medications are present:

Adult lumbar puncture (LP) kit (supplied per institution)

BD (Becton Dickinson) 22 or 25 gauge×3-7″ spinal needle (Quincke bevel)

Coaxial introducer needle (e.g., 18 G×3.5″), used at the discretion ofthe interventionalist (for introduction of spinal needle)

4 way small bore stopcock with swivel (Spin) male luer lock

T-connector extension set (tubing) with female luer lock adapter,approximate length 6.7″

Omnipaque 180 (iohexol), for intrathecal administration

Iodinated contrast for intravenous (IV) administration

1% lidocaine solution for injection (if not supplied in adult LP kit)

Prefilled 10 cc normal saline (sterile) flush syringe

Radiopaque marker(s)

Surgical prep equipment/shaving razor

Pillows/supports to allow proper positioning of intubated subject

Endotracheal intubation equipment, general anesthesia machine andmechanical ventilator

Intraoperative neurophysiological monitoring (IONM) equipment (andrequired personnel)

10 cc syringe containing AAV9.hIDUA vector; prepared and transported toCT/Operating Room (OR) suite in accordance with separate Pharmacy Manual

4. Day 1: Subject Preparation & Dosing

Informed Consent for the study and procedure are confirmed anddocumented within the medical record and/or study file. Separate consentfor the procedure from radiology and anesthesiology staff is obtained asper institutional requirements.

Study subject has intravenous access placed within the appropriatehospital care unit according to institutional guidelines (e.g., two IVaccess sites). Intravenous fluids are administered at the discretion ofthe anesthesiologist.

At the discretion of the anesthesiologist and per institutionalguidelines, study subject is induced and undergo endotracheal intubationwith administration of general anesthesia in an appropriate patient careunit, holding area or the surgical/CT procedure suite.

A lumbar puncture is performed, first to remove 5 cc of cerebrospinalfluid (CSF) and subsequently to inject contrast (Omnipaque 180)intrathecally to aid visualization of the cisterna magna. Appropriatesubject positioning maneuvers are performed to facilitate diffusion ofcontrast into the cisterna magna.

If not already done so, intraoperative neurophysiological monitoring(IONM) equipment is attached to subject.

Subject is placed onto the CT scanner table in the prone or lateraldecubitus position.

If deemed appropriate, subject is positioned in a manner that providesneck flexion to the degree determined to be safe during pre-operativeevaluation and with normal neurophysiologic monitor signals documentedafter positioning.

The following study staff and investigator(s) is (are) confirmed to bepresent and identified on-site:

-   -   Interventionalist/neurosurgeon performing the procedure    -   Anesthesiologist and respiratory technician(s)    -   Nurses and physician assistants    -   CT (or OR) technicians    -   Neurophysiology technician    -   Site Research Coordinator

The subject's skin under the skull base is shaved as appropriate.

CT scout images are performed, followed by a pre-procedure planning CTwith IV contrast, if deemed necessary by the interventionalist tolocalize the target location and to image vasculature.

Once the target site (cisterna magna) is identified and needletrajectory planned, the skin is prepped and draped using steriletechnique as per institutional guidelines.

A radiopaque marker is placed on the target skin location as indicatedby the interventionalist.

The skin under the marker is anesthetized via infiltration with 1%lidocaine.

A 22 G or 25 G spinal needle is advanced towards the cisterna magna,with the option to use a coaxial introducer needle.

After needle advancement, CT images are obtained using the thinnest CTslice thickness feasible using institutional equipment (ideally ≤2.5mm). Serial CT images should use the lowest radiation dose possible thatallows for adequate visualization of the needle and relevant softtissues (e.g., paraspinal muscles, bone, brainstem, and spinal cord).

Correct needle placement is confirmed by observation of CSF in theneedle hub and visualization of needle tip within the cisterna magna.

The interventionalist confirms the syringe containing vector ispositioned close to, but outside of the sterile field. Prior to handlingor administering vector, site confirms gloves, mask, and eye protectionare donned by staff assisting the procedure within the sterile field(other staff outside of sterile field do not need to take theseprocedures).

The short (˜6″) extension tubing is attached to the inserted spinalneedle, which is then attached to the 4-way stop cock. Once thisapparatus is “self-primed” with the subject's CSF, the 10 cc prefillednormal saline flush syringe will be attached to the 4-way stop cock.

The syringe containing vector is handed to the interventionalist andattached to a port on the 4-way stop cock.

Once the stop cock port to the syringe containing vector is opened, thesyringe contents are to be injected slowly (over approximately 1-2minutes), with care taken not to apply excessive force onto the plungerduring the injection.

Once the contents of the syringe containing AAV9.hIDUA are injected, thestop cock is turned so that the stopcock and needle assembly can beflushed with 1-2 cc of normal saline using the attached prefilledsyringe.

When ready, the interventionist alerts staff that he/she will remove theapparatus from the subject.

In a single motion, the needle, extension tubing, stopcock, and syringesare slowly removed from the subject and placed onto a surgical tray fordiscarding into a biohazard waste receptacle or hard container (for theneedle).

The needle insertion site is examined for signs of bleeding or CSFleakage and treated as indicated by the investigator. Site is dressedusing gauze, surgical tape and/or Tegaderm dressing, as indicated.

Subject is removed from the CT scanner and placed supine onto astretcher.

Anesthesia is discontinued and subject cared for following institutionalguidelines for post-anesthesia care. Neurophysiologic monitors can beremoved from study subject.

The head of the stretcher on which the subject lies should be slightlyraised (˜30 degrees) during recovery.

Subject is transported to a suitable post-anesthesia care unit as perinstitutional guidelines.

After subject has adequately recovered consciousness and is in stablecondition, he/she is admitted to the appropriate floor/unit for protocolmandated assessments. Neurological assessments are followed as per theprotocol and the Primary Investigator oversees subject care incollaboration with hospital and research staff.

Example 8: Evaluation of Intrathecal Routes of Administration in LargeAnimals

The purpose of this study was to evaluate more routine methods ofadministration into the CSF, including intraventricular (ICV) injection,and injection through a lumbar puncture. In brief, in this study ICV andIC AAV administration were compared in dogs. Vector administration wasevaluated via lumbar puncture in nonhuman primates with some animalsplaced in Trendelenburg position after injection, a maneuver which hasbeen suggested to improve cranial distribution of vector. In the dogstudy, ICV and IC vector administration resulted in similarly efficienttransduction throughout brain and spinal cord. However, animals in theICV cohort developed encephalitis, apparently due to a severe T cellresponse to the transgene product. The occurrence of thistransgene-specific immune response only in the ICV cohort is suspectedto be related to the presence of localized inflammation from theinjection procedure at the site of transgene expression. In thenon-human primate (NHP) study, transduction efficiency following vectoradministration into the lumbar cistern was improved compared to ourprevious studies by using an extremely large injection volume(approximately 40% of total CSF volume). However, this approach wasstill less efficient than IC administration. Positioning animals inTrendelenburg after injection provided no additional benefit. However,it was found that large injection volumes could improve cranialdistribution of the vector.

To maximize the effectiveness of intrathecal AAV delivery, it will becritical to determine the optimal route of vector administration intothe CSF. We previously reported that vector injection into the cisternamagna (cerebellomedullary cistern) by suboccipital puncture achievedeffective vector distribution in nonhuman primates, whereas injectionvia lumbar puncture resulted in substantially lower transduction of thespinal cord and virtually no distribution to the brain, underscoring theimportance of the route of administration [Hinderer, MolecularTherapy—Methods & Clinical Development. 12/10/online 2014; 1]. Othershave suggested that vector delivery into the lateral ventricles, acommon clinical procedure, results in effective vector distribution[Haurigot et al, J Clin Invest., August 2013; 123(8): 3254-3271]. It hasalso been reported that delivery via lumbar puncture can be improved byplacing animals in the Trendelenburg position after injection to promotecranial vector distribution [Meyer et al, Molecular therapy: the journalof the American Society of Gene Therapy. Oct. 31 2014]. In this study wecompared intraventricular and intracisternal administration of an AAV9vector expressing a green fluorescent protein (GFP) reporter gene indogs. We found that both routes achieve effective distributionthroughout the CNS, though intraventricular delivery may carryadditional risks of a transgene-specific immune response. We alsoevaluated vector delivery by lumbar puncture in NHPs, and the impact ofplacing animals in the Trendelenburg position after injection. There wasno clear effect of post-injection positioning, although we did find thatlarge injection volumes could improve cranial distribution of thevector.

A. Materials and Methods:

1. Vector production: The GFP vector consisted of an AAV serotype 9capsid carrying an expression cassette comprising a chicken beta actinpromoter with cytomegalovirus immediate early enhancer, an artificialintron, the enhanced green fluorescent protein cDNA, a woodchuckhepatitis virus posttranscriptional regulatory element, and a rabbitbeta globin polyadenylation sequence. The GUSB vector consisted of anAAV serotype 9 capsid carrying an expression cassette comprising achicken beta actin promoter with cytomegalovirus immediate earlyenhancer, an artificial intron, the canine GUSB cDNA, and a rabbit betaglobin polyadenylation sequence. The vectors were produced by tripletransfection of HEK 293 cells and purified on an iodixanol gradient aspreviously described [Lock et al, Human gene therapy. October 2010;21(10):1259-1271].

2. Animal experiments: All dogs were raised in the National ReferralCenter for Animal Models of Human Genetic Disease of the School ofVeterinary Medicine of the University of Pennsylvania (NIH ODP40-010939) under National Institutes of Health and USDA guidelines forthe care and use of animals in research.

3. NHP study: This study included 6 cynomolgus monkeys between 9 and 12years of age. Animals were between 4 and 8 kg at the time of injection.The vector (2×10¹³ GC) was diluted in 5 mL of Omnipaque (Iohexol) 180contrast material prior to injection. Injection of the vector via lumbarpuncture was performed as previously described [Hinderer, MolecularTherapy—Methods & Clinical Development. 12/10/online 2014; 1]. Correctinjection into the intrathecal space was verified by fluoroscopy. Foranimals in the Trendelenburg group, the head of the bed was lowered 30degrees for 10 minutes immediately following injection. Euthanasia andtissue collection were performed as previously described [Hinderer,Molecular Therapy—Methods & Clinical Development. 12/10/online 2014; 1].

4. Dog study: This study included 6 one-year-old MPS I dogs, as well asa 2 month old MPS VII dog. Baseline MRIs were performed on all ICVtreated dogs to plan the injection coordinates. Intracisternal injectionwas performed as previously described [Hinderer et al, Moleculartherapy: the journal of the American Society of Gene Therapy. August2015; 23(8):1298-1307]. For ICV injection, dogs were anesthetized withintravenous propofol, endotracheally intubated, maintained underanesthesia with isoflurane and placed in a stereotaxic frame. The skinwas sterilely prepped, and an incision was made over the injection site.A single burr hole was drilled at the injection site, through which a26-gauge needle was advanced to the predetermined depth. Placement wasconfirmed by CSF return. The vector (1.8×10¹³ GC in 1 mL) was slowlyinfused over one to two minutes. Euthanasia and tissue collection wereperformed as previously described [Hinderer et al, Molecular therapy:the journal of the American Society of Gene Therapy. August 2015;23(8):1298-1307].

5. Histology: Brains were processed as described for evaluation of GFPexpression [Hinderer, Molecular Therapy—Methods & Clinical Development.12/10/online 2014; 1]. GUSB enzyme stains and GM3 stains were performedas previously described [Gorda et al, Molecular therapy: the journal ofthe American Society of Gene Therapy. Oct. 8 2015.]

6. ELISPOT: At the time of necropsy blood was collected from vectortreated dogs in heparinized tubes. Peripheral blood mononuclear cellswere isolated by Ficoll gradient centrifugation. T cell responses toAAV9 capsid peptides and GFP peptides were evaluated by interferon gammaELISPOT. AAV9 and GFP peptide libraries were synthesized as 15-mers with10 amino acid overlap (Mimotopes). The AAV9 peptide library was groupedin 3 pools: Pool A from peptide 1 to 50, Pool B from peptide 51 to 100and Pool C from peptide 101 to 146. The GFP peptide library was alsogrouped in 3 pools. Phorbol 12-myristate 13-acetate plus Ionomycin salt(PMA+ION) were used as positive control. DMSO was used as negativecontrol. Cells were stimulated with peptide and interferon gammasecretion was detected as described. A response was considered positiveif it was both greater than 55 Spots Forming Units (SFU) per millionlymphocytes and at least 3 times the DMSO negative control value.

7. Biodistribution: At the time of necropsy tissues for biodistributionwere immediately frozen on dry ice. DNA isolation and quantification ofvector genomes by TaqMan PCR was performed as described [Wang et al,Human gene therapy. November 2011; 22(11):1389-1401].

8. GUSB enzyme assay: GUSB activity was measured in CSF as described[Gorda et al, Molecular therapy: the journal of the American Society ofGene Therapy. Oct. 8 2015].

B. Results

1. Comparison of Intraventricular and Intracisternal Vector Delivery inDogs

Our previous studies using a canine model of the lysosomal storagedisease mucopolysaccharidosis type I (MPS I) demonstrated that AAV9injection into the cisterna magna could effectively target the entirebrain and spinal cord [Hinderer et al, Molecular therapy: the journal ofthe American Society of Gene Therapy. August 2015; 23(8):1298-1307]. Inthis study, we compared distribution of an AAV9 vector expressing a GFPreporter gene administered into the cisterna magna or lateral ventricleof adult MPS I dogs. Three dogs were treated with a single 1 mLinjection of the vector (1.8×10¹³ genome copies) into the cisternamagna. Three additional dogs received a single vector injection of thesame vector into the lateral ventricle. For dogs treated by ICVinjection, a baseline MRI was performed to select the larger lateralventricle for injection and to define the target coordinates. Injectionwas performed using a stereotaxic frame to accurately target thedesignated ventricle.

The three dogs treated with IC vector injection appeared healthythroughout the study. They were euthanized two weeks after vectorinjection for evaluation of vector biodistribution and transgeneexpression. No gross or microscopic brain lesions were observed in anyIC treated dogs (FIGS. 12A-12F). Measurement of vector genomes byquantitative PCR revealed vector deposition throughout all sampledregions of the brain and spinal cord (FIG. 13). Consistent with thedistribution of vector genomes, robust transgene expression wasdetectable in most regions of cerebral cortex, as well as throughout thespinal cord (FIGS. 14A-14H). Spinal cord histology was notable forstrong transduction of alpha motor neurons, with a gradient oftransduction favoring thoracic and lumbar segments.

The three dogs treated with vector injected ICV initially appearedhealthy following the procedure. However, one animal (I-567) was founddead 12 days after injection. The other two animals survived to thedesignated 14 day necropsy time point, although one animal (I-565)became stuporous prior to euthanasia, and the other (I-568) began toexhibit weakness of facial muscles. These clinical findings correlatedwith significant gross brain lesions (FIGS. 12A-12F). Brains from allthree animals exhibited discoloration surrounding the needle track, withassociated hemorrhage in the animal that was found dead. Histologicalevaluation revealed severe lymphocytic inflammation in the regionsurrounding the injection site. Perivascular lymphocytic infiltrationwas also observed throughout the brain of each animal (FIGS. 12G and12H). Given this evidence for immunological toxicity, T cell responsesto both the AAV9 capsid protein and the GFP transgene were evaluated inperipheral blood samples collected from one of the ICV-treated dogs(I-565) at the time of necropsy. An interferon gamma ELISPOT showed astrong T cell response directed against GFP, with no evidence of aresponse to capsid peptides (FIG. 12I). This suggests that theencephalitis observed was caused by a cell-mediated immune responseagainst the transgene product.

Vector distribution in the ICV treated animals was similar to thatobserved in the IC treated group, although spinal cord transduction wassomewhat greater in the IC cohort (FIG. 13). GFP expression was observedthroughout the CNS regions examined in the ICV treated animals (FIGS.14A to 14H).

2. Impact of the Trendelenburg Position on CNS Transduction after AAV9Administration by Lumbar Puncture in NHP

We previously compared AAV9 injection into the cisterna magna or lumbarcistern of NHPs and found that the lumbar route was 10-fold lessefficient for targeting the spinal cord and 100-fold less efficient fortargeting the brain [C. Hinderer, et al, Molecular Therapy—Methods &Clinical Development. 12/10/online 2014; 1]. Other investigators havesince demonstrated better transduction using AAV9 administration bylumbar puncture, with improvements in cranial distribution of the vectorachieved by placing animals in the Trendelenburg position afterinjection [Myer et al, Molecular therapy: the journal of the AmericanSociety of Gene Therapy. Oct. 31 2014]. In this approach the vector wasdiluted into an excess volume of contrast material to increase thedensity of the solution and promote gravity driven distribution while inTrendelenburg. Six adult cynomolgus monkeys were treated with a singleinjection of AAV9 expressing GFP (2×10¹³ genome copies) in the L3-4interspace. The vector was diluted to a final volume of 5 mL in Iohexol180 contrast material. Four of the animals were positioned with the headof the procedure table at a −30° angle for 10 minutes immediately afterinjection. After 10 minutes fluoroscopic images were captured to verifycontrast distribution in the CSF. Notably with this large injectionvolume (approximately 40% of the total CSF volume of theanimal)[Reiselbach et al, New England Journal of Medicine. 1962;267(25):1273-1278] contrast material was rapidly distributed along theentire spinal subarachnoid space and into the basal cisterns even inanimals that were not placed in Trendelenburg position (FIGS. 16A and16B). Analysis of vector genome distribution by PCR (FIG. 17) and GFPexpression (FIGS. 18A to 18H) demonstrated transduction throughout thebrain and spinal cord. There was no apparent impact of post-injectionpositioning on the number or distribution of transduced cells. Aspreviously reported, there was vector escape to the periphery andhepatic transduction after intrathecal AAV administration [Hinderer etal, Molecular Therapy—Methods & Clinical Development. 12/10/online 2014;1; Haurigot et al, Journal of Clinical Investigation. August 2013;123(8):3254-3271]. The extent of liver transduction was dependent on thepresence of pre-existing neutralizing antibodies (nAb) against AAV9.Four out of six animals had no detectable baseline AAV9 nAbs (titer<1:5) and two animals (4051 and 07-11) had detectable pre-existingantibodies to AAV9 with a titer of 1:40. Consistent with previousresults, pre-existing antibodies blocked liver transduction, andresulted in increased vector distribution to the spleen [Wang et al,Human gene therapy. November 2011; 22(11):1389-1401, but had no impacton CNS transduction; Haurigot et al, Journal of Clinical Investigation.August 2013; 123(8):3254-3271].

C. Discussion

Because suboccipital puncture is not a common procedure in clinicalpractice, we evaluated more routine sites of CSF access, including thelateral ventricle and the lumbar cistern. Here we evaluated a methodemploying vector solutions with higher density and post-injectionTrendelenburg positioning to improve vector distribution cranially fromthe lumbar region.

In the dog study, both IC and ICV vector injection yielded similarlyeffective vector distribution, but encephalitis occurred only in the ICVgroup. A T cell response against the GFP transgene was detectable in oneof the ICV treated dogs, suggesting that the lymphocytic encephalitisobserved in these animals was due to a transgene-specific immuneresponse. Induction of a T cell response to a new antigen requires twoelements—recognition of an epitope from the protein by a naïve T cell,and an inflammatory “danger signal” that promotes activation of the Tcell. AAV is believed to be capable of expressing foreign transgeneswithout eliciting immunity against the transgene product because it doesnot activate the innate immune system, thereby avoiding inflammatorysignals and promoting tolerance rather than immunity when naïvelymphocytes encounter the newly expressed antigen. Local inflammationcaused by the trauma of penetrating the brain parenchyma, occurring atthe same location that the foreign transgene product is expressed, mayprovide the danger signal needed to induce an immune response to thetransgene product. This is supported by previous studies in MPS I dogs,which develop cell-mediated immune responses to an enzyme expressed froman AAV vector delivered by direct brain injection but not by ICinjection [Ciron et al, Annals of Neurology. August 2006; 60(2):204-213;Hinderer, et al, Molecular therapy: the journal of the American Societyof Gene Therapy. August 2015; 23(8):1298-1307]. The potential for suchan immune response depends on whether the transgene product isrecognized as foreign—for delivery of vectors expressing a protein thatis also produced endogenously, even an inflammatory response caused byinjection may not break tolerance to the self-protein. The results inthe study of ICV vector delivery in the MPS VII dog support thisconcept, as the similarity of the transgene product to an endogenousprotein was likely responsible for the absence of the type of T cellresponse that was observed to GFP. The same may be true for patientswith recessive diseases who carry missense mutations that allow forproduction of a protein similar to the transgene product. Risk ofimmunity could, therefore, vary depending on patient population andtransgene product, and in some cases immunosuppression may be necessaryto prevent destructive T cell responses to a transgene. The presentfindings suggest that the risk of deleterious immune responses canlikely be mitigated by using an IC rather than ICV route ofadministration.

The study of AAV9 administration via lumbar puncture in NHPs showedgreater transduction throughout the CNS than we have previously observedwith this route of administration. This difference appears to be due tothe large injection volume in the present study, which was necessary inorder to dilute the vector into an excess volume of contrast material.Previous studies have shown that such large volume injections(approximately 40% of CSF volume) can drive injected material directlyinto the basal cisterns and even the ventricular CSF of macaques[Reiselbach, cited above]. The potential to translate this approach tohumans is unclear, given that replicating this approach would requireextremely large injection volumes (>60 mL) that are not routinelyadministered to patients. Moreover, even with this high volume approach,injection via lumbar puncture was less efficient than previous resultswith IC delivery. In this previous study, animals were dosed by weight,so only one animal received an IC vector dose equivalent to that usedhere [Hinderer, et al, Molecular Therapy—Methods & Clinical Development.12/10/online 2014; 1]. That animal had on average 3-fold higher vectordistribution in the brain and spinal cord, indicating that even verylarge volume vector delivery to the lumbar cistern is less efficientthan IC delivery. In contrast to reports in the literature, we found noadditional benefit to placing animals in the Trendelenburg positionafter lumbar vector injection [Meyer et al, Molecular therapy: thejournal of the American Society of Gene Therapy. Oct. 31 2014].

Together these findings support vector administration at the level ofthe cisterna magna, as this approach achieves more efficient vectordistribution than administration via lumbar puncture, and appears tocarry less risk of immunity to the transgene product than ICVadministration. Vector delivery to the cisterna magna could be carriedout clinically using the suboccipital puncture approach that was used inpreclinical studies. Additionally, injection into the subarachnoid spacebetween the first and second cervical vertebra using a lateral approach(C1-2 puncture) is likely to produce similar vector distribution giventhe proximity of the injection site to the cisterna magna. The C1-2approach has the additional advantage that, unlike suboccipitalpuncture, it is widely used clinically for CSF access, particularly forintrathecal contrast administration.

Example 9: Non-Clinical Pharmacology/Toxicology Study ofAAV2/9.CB7.CI.hIDS.RBG Injected Intrathecally in Rhesus Macaques

A study designed to evaluate the safety of intrathecal administration oftwo doses of AAV2/9.CB7.CI.hIDS.RBG, a vector encoding human IDS, inrhesus macaques is performed, to obtain an adequate safety margin abovethe Minimum Efficacious Dose (MED) for human dosing.

Control article is administered via suboccipital puncture to a singlemacaque randomized to Group 1. Test Article is administered viasuboccipital puncture to 6 rhesus macaques randomized to Groups 2-3.Macaques in Group 2 receive test article at a high dose of 5×10¹³ GenomeCopy (GC) (N=3); macaques in Group 3 receive test article administeredat a low dose of 1.7×10¹³ GC (N=3). Blood and cerebrospinal fluid arecollected as part of a general safety panel.

Following completion of the in-life phase of these studies at 90±3 dayspost-vector administration, macaques are necropsied with tissuesharvested for a comprehensive histopathological examination. Lymphocytesare harvested from the liver, spleen, and bone marrow to examine thepresence of cytotoxic T lymphocytes (CTLs) in these organs at the timeof necropsy.

I. Experimental Design

A. Materials

Test article—AAV9.CB7.hIDS is also known as

AAV2/9.CB7.CI.hIDS.RBG, also known as AAV2/9.CB7.CI.hIDS.RBG.KanR. Thesedesignations are synonymous and may be used interchangeably.

The test article was produced as a single lot with a concentration of5.72×10¹³ GC/ml measured by Droplet digital (dd) PCR is used. Vectorsare stored at ≤−60° C. after production until the day of injection. Onthe day of injection, vector is diluted with the control article. Oncediluted, vector is stored at 2-4° C. in a refrigerator or on wet iceuntil the time of injection. Vector preparations are performed on theday of injection.

Control group animals are administered with the control article Elliot'sFormulation Buffer (EFB) and no test article (EFB+0.001% Pluronic F68).Control article is stored at room temperature after production and untilthe day of injection.

B. Test System

Justification for Test System Selection: This study involves intrathecal(IT) delivery of a gene therapy vector for CNS diseases. The dimensionsof the CNS in the non-human primate (NHP) acts as the bestrepresentative model of our clinical target population. This studyprovides data about dose-related toxicity of the vector after ITinjection.

Seven (7) 3-7 year old male Macaca mulatta (Rhesus macaques) with aweight between 3 to 10 kg supplied by Covance Research Products, Inc.(Alice, Tex.) are used.

II. General Design Procedures

7 rhesus macaques (males) are used in the study Animals are divided intothree study groups as listed in Table 1. All 7 animals receive an ITinjection via suboccipital puncture.

TABLE 1 Group Designations Group designation 1 2 3 Number of animals 1 33 per group Gender M M M Treatment control AAV9.CB7.HIDS AAV9.CB7.HIDSarticle high dose low dose 1 mL 5 × 10¹³ GC 1.7 × 10¹³ GC in 1 mL in 1mL Necropsy day 90  90  90 

Animals are anesthetized and dosed with test article via sub-occipitalpuncture into the cisterna magna.

Anesthetized macaques are prepped in a procedure room and transferred tothe Fluoroscopy Suite and placed on an imaging table in the lateraldecubitus position with the head flexed forward for CSF collection anddosing into the cisterna magna. The site of injection is asepticallyprepared. Using aseptic technique, a 21-27 gauge, 1-1.5 inch Quinckespinal needle (Becton Dickinson) is advanced into the sub occipitalspace until the flow of CSF is observed. Up to 1.0 mL of CSF iscollected for baseline analysis and prior to dosing. The anatomicalstructures that are traversed include the skin, subcutaneous fat,epidural space, dura and atlanto-occipital fascia. The needle isdirected at the wider superior gap of the cisterna magna to avoid bloodcontamination and potential brainstem injury. Correct placement ofneedle puncture is verified via myelography, using a fluoroscope(OEC9800 C-Arm, GE). The fluoroscope is operated in accordance withmanufacturer recommendations. After CSF collection, a leur accessextension catheter is connected to the spinal needle to facilitatedosing of Iohexal (Trade Name: Omnipaque 180 mg/mL, General ElectricHealthcare) contrast media and test or control article. One (1) mL ofIohexol is administered via the catheter and spinal needle. Afterverifying needle placement, a syringe containing the test article(volume equivalent to 1 mL plus the volume of syringe and linker deadspace) is connected to the flexible linker and slowly injected over20-60 seconds. The needle is removed and direct pressure applied to thepuncture site. Residual test article remaining in the injectionapparatus is collected and stored at ≤−60° C.

In the event that an animal cannot be dosed successfully to the cisternamagna, dosing into the C1-C2 intrathecal space (atlanto-axial joint) maybe used as an alternate site of dosing. Dosing procedure is as follows.The animal is placed in the lateral decubitus position with the headflexed forward for CSF collection and dosing into the C1-C2 intrathecalspace. The site of injection is aseptically prepared. Using aseptictechniques, a 21-27 gauge, 1-1.5 inch Quincke spinal needle (BectonDickinson) is advanced into the intrathecal space until the flow of CSFis observed. Up to 1.0 mL of CSF is collected for baseline analysis andprior to dosing. The anatomical structures that is traversed include theskin, subcutaneous fat, epidural space, dura and fascia. The needle isinserted superior to C2 vertebra and into the vertebral space as toavoid blood contamination and potential injury to the cervical spinalcord. The rest of the procedure is similar to that previously describedfor cisterna magna administration.

The animals receive an intrathecal injection of either AAV9.CB7.hIDS orcontrol article. The frequency of dosing is outlined in Table 3. Thehigh dose of AAV9.CB7.hIDS is 5×10¹³ GC and the low dose 1.7×10¹³ GC.The total volume of diluted AAV9.CB7.hIDS or control article to beinjected per macaque is 1 mL.

III. Results

CSF pleocytosis was observed in 2 out of 3 animals in the high-dosetreated group (Group 2) and 1 out of 3 animals in the low-dose treatedgroup (Group 3) shown as white blood cell count in CSF (FIG. 28). Suchpleocytosis was resolved on Day 90 in all animals but one high-dosetreated animal (RA 2203).

In addition, dorsal columns axonopathy is present (data not shown).

Current low dose corresponds to 1.9×10¹¹ GC/g brain weight, which isclose to the proposed clinical high dose, 9.4×10¹⁰ GC/g brain weight.Current high dose corresponds to 5.6×10¹¹ GC/g brain weight, which isapproximately 5 times of the clinical dose.

ELISA assay of anti-hIDS antibody was performed on the serum samplescollected from the animals described above. Results obtained on Day 60were plotted in FIG. 29 and shown in Table 2 below. CSF samples werediluted 20 times for evaluation via ELISA assay of anti-hIDS antibody.The result is shown in Table 3. Decreased immunogenicity was observed inthe low-dose treated group (Group 2) compared to the high-dose one(Group 3) in both serum and CSF. No immunogenicity against hIDS wasobserved in the non-treated control.

TABLE 2 anti hIDS antibody ELISA (Serum) Titer RA2198 vehicle <50 RA2197low dose 400 RA 2203 high dose 1600 RS 2231 high dose 6400

TABLE 3 anti hIDS antibody ELISA (CSF) CSF 1:20 RA2198 vehicle0.050066667 RA2197 low dose 0.052133333 RA 2203 high dose 0.058833333 RS2231 high dose 0.141566667

Example 10: Non-Clinical Pharmacology/Toxicology Study of AAV9.CB7.hIDSInjected Intrathecally in Immunosuppressed Rhesus Macaques

A study designed to evaluate the impact of chemically inducedimmunosuppression (IS) on the safety of intrathecal administration oftwo doses of AAV9.CB7.hIDS, a vector encoding human IDS, in rhesusmacaques, is performed. The study is as described in Example 9 withmodifications noted below.

Control article dosed animal are repeated as in Example 9. Test Articleis administered via suboccipital puncture to 6 rhesus macaquesrandomized to Groups 1-2. Macaques in Group 1 receive test article at ahigh dose of 5×10¹³ Genome Copy (GC) (N=3); macaques in Group 2 receivetest article administered at a low dose of 1.7×10¹³ GC (N=3). Blood andcerebrospinal fluid are collected as part of a general safety panel.Monkeys from group 1 and 2 receive mycofenolate mofetil (MMF) at least 2weeks prior to AAV9.CB7.hIDS dosing and up to and including day 60 afterdosing, and rapamycin at least 2 weeks prior to dosing and up to andincluding day 90 (+/−3 days) after dosing. Plasma trough levels for bothimmunosuppressive drugs are monitored and doses adjusted to maintain arange of 2-3.5 mg/L for mycofenolate acid (MPA, active metabolite ofMMF) and 10-15 μg/L for rapamycin.

Following completion of the in-life phase of these studies at 90±3 dayspost-vector administration, macaques are necropsied with tissuesharvested for a comprehensive histopathological examination. Lymphocytesare harvested from the blood, spleen, and bone marrow to examine thepresence of cytotoxic T lymphocytes (CTLs) in these tissues at the timeof necropsy.

A. Materials

The test article, control article, preparation thereof is described inExample 9.

B. Test System

Macaca mulatta (Rhesus macaques) is utilized and kept as described inExample 9. A cohort of 6 animals (6 males) is used for the study.Animals are assigned a number from 1-6 from the lowest ID to thehighest. A random list of numbers 1-6 is generated by random.org andonce randomized the animals are assigned in order as follows: Group 1 isassigned three animals; and Group 2 is assigned three animals.

C. General Design Procedures

Sample Size and Group Designations:

6 rhesus macaques are used in the study. Animals are divided into 2study groups as listed in Table 4. All 6 animals receive an IT injectionvia suboccipital puncture

TABLE 4 Group Designations Group designation 1 2 Number of animals 3 3per group Gender M M Treatment AAV9.CB7.hIDS AAV9.CB7.hIDS high dose lowdose 5 × 10¹³ GC 1.7 × 10¹³ GC in 1 mL in 1 mL Immunosuppression Yes YesNecropsy day 90  90 

An immunosuppression regimen is administered to all animals in Groups 1and 2. Drug combinations, dose, and administration schedule for eachgroup of animals is summarized in Table 5 Animals may be treated withsystemic antibiotics and/or antifungals to treat opportunisticinfections associated with immunosuppression if they occur.

Animals are dosed with a combination therapy of Mycophenolate mofetil(MMF) and Rapamycin. Both immunosuppression drugs are administeredthrough an orogastric feeding tube or nasogastric feeding tube toanesthetized or conscious chair restrained macaques. Anesthetizedanimals are not be fasted to allow aspiration of gastric content fromthe tube (correct placement verification). The immunosuppression regimenstart at least two weeks prior to intrathecal dosing of the testarticle. Starting doses of IS drugs are based upon efficacy previouslydescribed in rhesus macaques, upon doses previously used at GTP forother studies, and are adjusted upon plasma trough level monitoring.

The weight of animals on the first day of immune suppression is used tocalculate the initial dose. Animals are then weighed every morning andthe dose recalculated if the weight change exceeds +/−10%.

Trough levels are monitored 2 times per week initially, and once a weekafter Day 60 (when rapamycin only will be administered).

TABLE 5 Immunosuppression Regimen MMF, oral administration Rapamycin,oral administration Dose/ Dose/ Group schedule Duration scheduleDuration 2 20-100 d −14 to d 60 0.5-4 d −14 to d 90 mg/kg BID mg/kg SID3 20-100 d −14 to d 60 0.5-4 d −14 to d 90 mg/kg BID mg/kg SID

c. IS Drugs

Rapamycin is administered at a dose of 0.5-4 mg/kg PO, SID a minimum of14 days prior to intrathecal dosing and up to and including Day 90 (+/−3days) of the study. Starting dose is 1 mg/kg. Rapamycin doses arecalculated to maintain target trough levels as close to 10-15 μg/L aspossible for the duration of the study. If the target trough drug levelsare not achieved within one week, the doses are titrated in 0.25-2 mg/kgdose intervals. After the stabilization period, if the trough levels aretoo low for 2 consecutive bleedings, an adjustment is made. If they aretoo high, immediate adjustment is made.

MMF is administered for a minimum of 14 days prior to intrathecal dosingand up to and including Day 60 of the study at a starting dose of 50mg/kg PO, BID. Trough levels results are then used to adjust the dosesthat are titrated in 5-25 mg/kg dose intervals. Trough levels for MPAare maintained as close to 2-3.5 mg/L as possible. After stabilizationperiod, if the trough levels are too low for 2 consecutive bleedings, anadjustment is made. If they are too high, immediate adjustment is made.

d. Formulation of IS Drugs

MMF is administered orally to the test system using a commerciallyavailable oral solution at a concentration of 200 mg/mL.

Rapamycin is administered orally to the test system using one or more ofthe following commercially available formulations: 0.5 mg coatedtablets; 1 mg coated tablet; 2 mg coated tablet.

When administering pill formulations, tablets are prepared as follows:

Each rapamycin pill is placed in a diluent of either room temperature orwarm water (warm water is used to accelerate the dissolution if needed)to dissolve the outer coating of the tablet. Approximately 10 ml ofwater is used. The pill is white under the yellow coating.

Once the outer coating is dissolved, each pill is crushed using a mortarand pestle until it is a fine powder, creating a uniform solution. Thetotal volume of the diluent used to dissolve the tablet(s) is recordedin the study record.

The total mixture is drawn into a dose syringe and administered througha OG or NG tube as previously described in this Example

Following dosing of MMF and/or Rapamycin the orogastric or nasogastricfeeding tube is flushed with drinking water. The volume of flush isrecorded in the study record.

The following studies were performed to evaluate the efficacy ofintracerebroventricular (ICV) delivery of AAV9.CB7.CI.hIDS in MPS IImice and to determine the minimum effective dose. Efficacy was based onevidence of a pharmacodynamic response to the vector (generation ofenzymatically active hIDS protein) and an effect on behavioral andhistological manifestations of MPS II disease (IDS deficiency).

Example 11: Efficacy of Intracerebroventricular AAV9.CB7.CI.hIDS.rBG ina Mouse Model of MPS II I. Summary

Hunter syndrome, mucopolysaccharidosis type II (MPS II), is a X-linkedinherited disorder caused by the deficiency of the enzymeiduronate-2-sulfatase (IDS), involved in the lysosomal catabolism of theglycosaminoglycans (GAG) dermatan and heparan sulfate. This deficiencyleads to the intracellular accumulation of undegraded GAG and eventuallyto a progressive severe clinical phenotype. Many attempts have been madein the last two to three decades to identify possible therapeuticstrategies for the disorder, including gene therapy and somatic celltherapy. Study was performed to evaluate the short-term (21 days)biodistribution, expression, and activity of a singleintracerebroventricular (ICV) administration of AAV9.CB7.CI.hIDS.rBG, anAAV9 vector expressing human IDS, in a murine model of MPS II. A furtherstudy was designed and performed to determine the minimum effective dose(MED) of AAV9.CB7.CI.hIDS.rBG administered through the ICV route as asingle dose with a 3 months post-injection (pi) observation period, in amurine model of MPS II. The study also included some safety endpoints(evaluation of the humoral immune response to the transgene and brainhistopathology).

AAV9.CB7.CI.hIDS.rBG was administered intracerebroventricularly to 2-3month old C57BL/6 IDS γ/− (MPS II) mice (16 males/group) at doses of3×10⁸ GC or 3×10⁹ GC or 3×10¹⁰ GC (determined by qPCR tittering of thevector) on Day 0. At Day 21, mice from groups 3-5 (Table 6) wereeuthanized and necropsied for evaluation of CNS IDS activity,biodistribution and anti-hIDS immunogenicity. Between Days 60 and 89,wildtype mice and untreated MPS II mice were evaluated in a series ofneurobehavioral assays (open field, Y-maze, contextual fear conditioningand novel object recognition) to characterize effect of the diseasestate on these endpoints. Based on the results of these initial assays,remaining treated mice were evaluated in the two assays that evaluatedlong term memory (contextual fear conditioning and novel objectrecognition). Approximately 3 months after ICV dosing ofAAV9.CB7.CI.hIDS.rBG, all the remaining mice were euthanized andnecropsied. Serum and CSF were evaluated for IDS activity as well asserum for anti-IDS antibodies. Liver and heart were evaluated for GAGtissue content. Brain overall lysosomal storage (both primary GAGstorage and secondary ganglioside storage) was assessed byimmunohistochemical staining of LIMP2 and GM3. Tissue hexosaminidaselevels have been shown to be higher in MPS II mice and MPS II patients,and to be a biomarker of lysosomal homeostasis disruption. Tissuehexosaminidase enzymatic activity was thus measured as a biomarker oflysosomal function secondary to AAV9.CB7.CI.hIDS.rBG administration.Histopathology of the brain was evaluated to investigate both efficacyand safety.

TABLE 6 Combined Study Design for Studies W2301 and W2356 N per Endpoint(Animal IDs) Study W2301 Study Neurobehavioral Dose W2356 Testing DaysGroup Dose Dose Volume Necropsy 60-89 Necropsy # Genotype (GC qPCR) (GCddPCR) (μL) Day 21 3 months pi t C57BL/6J 0 0 0 3* (351, 8 (33, 36,I2S^(γ/+) (untreated) (untreated) 353, 365) 43, 44, 45, (Wildtype) 64,78, 79) 2 C57BL/6J 0 0 0 5* (157, 8 (27, 34, I2S^(γ/−) (untreated)(untreated) 194, 201, 35, 42, 46, (MPS II) 258, 276) 57, 71, 72) 3C57BL/6J 3e8 5.2e8 5 8 (158, 8 (84‡, 89, I2S^(γ/−) 166, 232, 100, 101,117, (MPS II) 233, 234, 140, 141, 142) 263, 265, 270) 4 C57BL/6J 3e95.2e9 5 8 (148, 8 (107, 108, I2S^(γ/−) 149, 187, 120, 121, 122, (MPS II)219, 220, 123, 136, 139) 244, 245, 247) 5 C57BL/6J 3e10 5.2e10  5 8(208, 8 (91, 93, I2S^(γ/−) 211, 212, 94, 95, 128, (MPS II) 251, 253,129, 130, 132) 267, 268, 269) *Control values for brain and CSF I2Sactivity were based on tissue samples collected from untreated wildtypeand MPS II animals that were taken from the breeding colony. ‡Animal 84was incorrectly genotyped as MPS II (I2Sγ/−) but proved on regenotypingto be wildtype (I2Sγ/+). Data from this animal was excluded from allanalyses.

ICV administration of AAV9.CB7.CI.hIDS.rBG to C57BL/6 IDS γ/− (MPS II)mice at up to 3×10¹⁰ GC (5.2×10¹⁰ GC by ddPCR method) was welltolerated, with no clinical signs or mortality, and resulted indistribution to the CNS as well as to peripheral tissues, particularlyliver, thus demonstrating partial redistribution of injected viralparticles from the CSF to the peripheral blood.

There was evidence of IDS gene expression in the brain as evidenced bydetection of the vector and dose dependent increases in IDS activity inthe brain at Day 21 and in the CSF 3 months pi, with enzymatic activityclose to wildtype level at the highest dose (brain tissue) andcomparable to higher than wildtype at the mid and high doses (CSF).There was dose dependent normalization of the lysosomal compartment, asshown by reductions in LIMP2 and GM3 staining in the CNS at all doses 3months pi. In H&E stained brain sections, dose dependent reductions inthe amount and frequency of glial vacuolation and neuronal accumulationof amphiphilic material, indicators of MPS II CNS phenotype, were alsoobserved. Corresponding to the changes in CNS lysosomal content andimprovements in disease-related morphology in the H&E stained sections,there were improvements in one measure of long term memory (novel objectrecognition, NOR) but not in the other, contextual fear conditioning(CFC). No clear dose response was apparent in the improvement in NOR.

Dose dependent increases in serum IDS activity were also observed 3months pi, with enzymatic activity comparable to or higher than wildtype at the mid and high doses. Reflecting the normalization of IDSactivity in serum, the treated MPS II mice had dose dependent decreasesin hexosaminidase activity and GAG content in the liver and heart.Hepatic Hexosaminidase and GAG levels were normalized at all dose levelsin lever and at the mid and high doses in the heart. The highlytransduced liver (1 to 10 GC per diploid genome) probably acted as adepot organ for the secretion of IDS in the serum and cross-correctionof the heart at the higher doses.

There was no evidence of test-article related toxicity in the brain,although changes related to the ICV administration procedure itself wereobserved in some mice. Humoral immune response to the transgene wasminor, observed only in some mid- and high-dose animals without impacton the health or brain histopathology of those animals.

In conclusion, AAV9.CB7.CI.IDS.rBG was well tolerated in MPS II mice atall dose levels and resulted in dose-dependent increases in IDS levels(expression and enzymatic activity) that were associated withimprovements in both CNS and peripheral parameters of MPS II. The lowestdose administered, 3×108 GC (5.2×10⁸ GC ddPCR method), was the minimumeffective dose in this study.

II. Materials and Methods

A. Test article identified as AAV9.CB7.CI.hIDS.rBG is clear, colorlessliquid with a titer at 1.18×10¹³ GC/mL measured by qPCR and 2.057×10¹³GV/mL measured by ddPCR. Endotoxin is <1.0 EU/mL. Purity is 100%. Thetest article was stored at ≤−60° C.

B. Dose Formulation and Analysis

1. Preparation of Test Article:

The test article was diluted with sterile phosphate buffered saline(PBS) to the appropriate concentration for each dose group. Dilutedvector was kept on wet ice and injected to the animals within 4 hoursafter dilution.

C. Test System

Mus musculus C57BL/6J IDSγ/− (MPS II phenotype, N=56 males) and C57BL/6JIDSγ/+ (Wild type phenotype, N=8 males) were bred at the TranslationalResearch Laboratories (TRL) Vivarium from stock originally obtained fromJackson Laboratories (Stock No. 024744). Animals were 2-3 months of ageat Day 0 (day of dosing).

D. Experimental Design

1. Study W2356

On day 0, all animals from groups 3-5 (16 males/group, W2356 and W2301studies combined) were dosed ICV with AAV9.CB7.CI.hIDS.rBG. On day 21,mice/group from groups 3-5 (8 males/group, W2356 study) were euthanizedand necropsied and brain, heart, lung, liver, and spleen were collectedand snap frozen on dry ice for an evaluation of brain IDS activity andtissue vector biodistribution.

TABLE 7 W2356 Study Design Dose N per Endpoint Group Dose Dose Volume(Animal IDs) # Genotype (GC qPCR) (GC ddPCR) (μL) NecropsyDay 21 1C57BL/6J IDS^(γ/+) 0 0 0 3* (351, 353, 365) (Wildtype) (untreated)(untreated) 2 C57BL/6J IDS^(γ/−) 0 0 0 5* (157, 194, 201, (MPS II)(untreated) (untreated) 258, 276) 3 C57BL/6J IDS^(γ/−) 3 × 10⁸ 5.2 × 10⁸5 8 (158, 166, 232, 233, (MPS II) 234, 263, 265, 270) 4 C57BL/6JIDS^(γ/−) 3 × 10⁹ 5.2 × 10⁹ 5 8 (148, 149, 187, 219, (MPS II) 220, 244,245, 247) 5 C57BL/6J IDS^(γ/−)  3 × 10¹⁰  5.2 × 10¹⁰ 5 8 (208, 211, 212,251, (MPS II) 253, 267, 268, 269) *Control values for brain and CSF IDSactivity were based on tissue samples collected from untreated wildtypeand MPS II animals that were taken from the breeding colony.

2. Study W2301

On Day 0, all animals from groups 3-5 (16 males/group) were dosed ICVwith AAV9.CB7.CI.hIDS.rBG. From 2 to 3 months pi: Animals from groups 1and 2 (8 mice/group) were evaluated in a series of neurobehavioralassays including open field activity, Y-maze activity, contextual fearconditioning, and novel object recognition to determine if there wereeffects of the disease state (MPS II genotype) on these endpoints. If anapparent effect of disease was observed, 8 mice/group from Groups 3-5were also evaluated in that assay to determine if treatment with thevector had an effect on response.

Animals from groups 1-5 (same animals evaluated in neurobehavioralassays, 8 mice/group) were deeply anesthetized (ketamine-xylazine) tocollect CSF (cisterna magna puncture) and blood (cardiac puncture) forevaluation of serum and CSF IDS activity and serum anti-hIDS antibodies.The mice were then euthanized and necropsied and samples were collectedfor brain histopathology; brain lysosomal storage (assessed byimmunohistochemistry and image analysis); liver and heart hexosaminidaseactivity; and liver and heart GAG content (assessed by tissue content).

TABLE 8 W2301 Study Design N per Endpoint (Animal IDs) NeurobehavioralDose Testing Group Dose Dose Volume Days 60-89 # Genotype (GC qPCR) (GCddPCR) (μL) Necropsy 3 months pi 1 C57BL/6J IDS^(γ/+) 0 0 0 8 (33, 36,43, 44, 45, (Wildtype) (untreated) (untreated) 64, 78, 79) 2 C57BL/6JIDS^(γ/−) 0 0 0 8 (27, 34, 35, 42, 46, (MPS II) (untreated) (untreated)57, 71, 72) 3 C57BL/6J IDS^(γ/−) 3 × 10⁸ 5.2 × 10⁸ 5 8 (84‡, 89, 100,101, (MPS II) 117, 140, 141, 142) 4 C57BL/6J IDS^(γ/−) 3 × 10⁹ 5.2 × 10⁹5 8 (107, 108, 120, 121, (MPS II) 122, 123, 136, 139) 5 C57BL/6JIDS^(γ/−)  3 × 10¹⁰  5.2 × 10¹⁰ 5 8 (91, 93, 94, 95, 128, (MPS II) 129,130, 132) ‡Animal 84 was incorrectly genotyped as MPS II (IDS γ/−) butproved on regenotyping to be wildtype (IDS γ/+). Data from this animalwas excluded from all analyses.

3. Administration of Test Article

The ICV route was chosen because it is minimally invasive and requiresno surgical procedure in the mouse (compared to the cisterna magna routethat necessitates neck skin and muscle incision). As this study includedsome neurobehavioral endpoints involving mice activity recording, anon-invasive injection that did not involve survival surgery waspreferred. It was demonstrated previously by us and others that In bothmice and large animals, a single injection of AAV9 into thecerebrospinal fluid (ICV or cisterna magna) targets neurons within thewhole CNS (Dirren at al., Hum. Gene. Therapy 25, 109-120 (2014); Snyderet al. Hum. Gene Ther 22, 1129-1135 (2011), Federici et al. Gene Therapy19, 852-859 (2012), Haurigot et al. J Clin Invest 123, 3254-3271 (2013),Bucher et al. Gene Therapy 21, 522-528 (2014), Hinderer et al. Mol Ther:22, 2018-2027 (2014) and Mol Ther—Methods & Clin Dev 1, 14051 (2014)].The data published by Haurigot et al. (2013) specifically addresses thecomparison between ICV and cisterna magna injection in the context ofanother lysosomal storage disease, demonstrating that the two routes ofadministration are equivalent in terms of transgene expression andbiodistribution.

The maximum feasible dose due to volume constraint (5 μL for ICVinjections in adult mice) was approximately 6×10¹⁰ GC per mouse (qPCRtittering method). Due to previous results obtained at GTP for thetreatment of other MPSs and due to the concerns regarding scalability tolarger animals, the vector was diluted in order to inject 3×10¹⁰, 3×10⁹,or 3×10⁸ GC per mouse (qPCR tittering method). Based on the ddPCRmethod, the actual doses were 5.2×10¹⁰, 5.2×10⁹, and 5.2×10⁸ GC permouse. Considering an average brain mass of 0.4 g in young adults C57B16mice (Biology of the laboratory mouse by the staff of the Jacksonlaboratory, second revised edition, Earl L. Green Editor), this isequivalent to 1.31×10¹¹ GC per gram of brain mass at the highest doseand 1.31×10⁹ GC per gram of brain mass at the lowest dose.

Vector was administered ICV into the right lateral ventricle. Mice wereanesthetized with Isoflurane. Each anesthetized mouse was grasped firmlyby the loose skin behind the head and injected free hand anterior andlateral to the bregma with a Hamilton syringe fitted with a 26-gaugeneedle, which was adjusted to be inserted 3 mm deep. The injectionmethod was previously validated in mice by injection of dye or substanceP into the right lateral ventricle. Success was defined as visualizationof blue dye or by itching behavior (after substance P administration) ofthe mice following injection into the cerebrospinal fluid.

F. Procedures, Observations and Measurements

Animals were monitored daily for morbidity/mortality. The animals weremonitored by daily cage-side visual observations for general appearance,signs of toxicity, distress and changes in behavior. These data were notrecorded in this non-GLP study.

Between Days 60 and 90 (2-3 months after vector administration), 8animals from Groups 1 and 2 of Study W2356 were subjected to behavioraland neurocognitive tests to determine effect of genotype on theseendpoints. If an effect of genotype was identified, additional testingwas done using 8 animals from Study W2356 Groups 3-5 to determine ifthere was an effect of treatment. All behavior procedures were performedby operators blinded to genotype and group.

a) General Locomotion (Open Field Activity):

Spontaneous activity in an open field was measured with a PhotobeamActivity System (PAS)—Open Field (San Diego Instruments). In thisassessment, mice were individually placed in the arena for a single10-minute trial. Horizontal and vertical beam breaks were collected toassess general locomotion and rearing activity.

b) Short Term Memory (Y Maze Activity):

Y Maze Spontaneous Alternation is a behavioral test for measuring thewillingness of rodents to explore new environments. Testing occurs in aY-shaped maze with three white, opaque plastic arms at a 120° angle fromeach other. After introduction to the center of the maze, the animal isallowed to freely explore the three arms. Rodents typically prefer toinvestigate a new arm of the maze rather than returning to one that waspreviously visited. Over the course of multiple arm entries, the subjectshould show a tendency to enter a less recently visited arm. The numberof arm entries and the number of triads are recorded in order tocalculate the percentage of alternation. An entry is defined as all fourlimbs present within the arm. This test is used to quantify cognitivedeficits in transgenic strains of mice and evaluate novel chemicalentities for their effects on cognition. Many parts of thebrain—including the hippocampus, septum, basal forebrain, and prefrontalcortex—are involved in this task. In this study, a standard Y-shapedmaze (San Diego Instruments) was used, and the sequence and number ofarm entries was recorded during an 8-minute trial. A spontaneousalternation (SA) was defined as sequential entry into all 3 arms of themaze without immediately returning to a previously entered arm. Totalarm entries (AE) as collected as a measure of motor activity. Thepercent spontaneous alternation was calculated as % SA=(SA/(AE−2)*100).

c) Long Term Memory (Contextual Fear Conditioning):

In these tasks, animals learn to fear a new environment or anemotionally neutral conditioned stimulus (CS), such as a tone, becauseof its temporal association with an aversive unconditioned stimulus(US), usually foot shock. When exposed to the same context or the sameCS, conditioned animals show freezing behavior (Abel et al, Cell 88:615-626. 1997). On the training day, mice were allowed to explore theunique conditioning chamber (Med Associates Inc) for 300 seconds.Between 248-250 seconds of the 300 second period, a non-signaled, 1.5 mAcontinuous foot shock was delivered. After an additional 30 seconds inthe chamber, the mice were returned to their home cage. Twenty fourhours later, recall of spatial context was assessed for 5 consecutiveminutes in the same chamber where training occurred. Memory was assessedwith software used to score freezing behavior (Freezescan, CleverSysInc). The percent freezing in the 2.5 minute prestimulus epoch of thetraining session (prior to the administration of the foot shock) iscompared to the percent freezing upon reexposure to the chamber. Anincrease in freezing indicates recall of the foot shock (i.e. thatlearning has occurred, and the animals associate the chamber with thefoot shock).

d) Long Term Memory (Novel Object Recognition):

The Novel Object Recognition (NOR) task is used to evaluate cognition,particularly recognition memory, in rodent models of CNS disorders. Thistest is based on the spontaneous tendency of rodents to spend more timeexploring a novel object than a familiar one. The choice to explore thenovel object reflects the use of learning and recognition memory. TheNovel Object Recognition task is conducted in an open field arena withtwo different kinds of objects that are generally consistent in heightand volume, but are different in shape and appearance. Duringhabituation, the animals are allowed to explore an empty arena.Twenty-four hours after habituation, the animals are exposed to thefamiliar arena with two identical objects placed at an equal distance.The next day, the mice are allowed to explore the open field in thepresence of the familiar object and a novel object to test long-termrecognition memory. The time spent exploring each object and thediscrimination index percentage are recorded. In this study, theexperimental apparatus consisted of a gray rectangular arena (60 cm×50cm×26 cm) on a white floor and the two unique objects were metal bars3.8×3.8×15 cm and PVC pipes 3.2 cm diameter×15 cm in length. During a5-day habituation phase, mice were handled 1-2 minutes/day and permittedto explore the empty arena for 5 minutes/day. During the training phase,two identical objects were placed in the arena and the mice werepermitted to explore the objects for 15 minutes. In the recall phase,mice were returned to the arena with one familiar object and one novelobject. Normal mice preferentially explore the novel object. Allsessions were recorded, and time spent exploring the objects was scoredwith an open source image analysis program [Patel et al Front BehavNeurosci 8:349 (2014)].

I. Laboratory Evaluations

1. IDS Activity in Serum and CSF

Blood for serum and CSF IDS activity was collected at necropsyapproximately 3 months post injection. Serum was separated from bloodand serum and CSF frozen on dry ice and stored at −80° C. untilanalyzed. IDS activity was measured by incubating 10 μL sample with 20μL of 1.25 mM 4-methylumbelliferyl (MU) a-L-idopyranosiduronic acid2-sulfate (Santa Cruz Biotechnology) dissolved in 0.1M sodium acetatewith 0.01M lead acetate, pH 5.0. After incubating 2 h at 37° C., 45 μLof McIlvain's buffer (0.4M sodium phosphate, 0.2M sodium citrate, pH4.5) and 5 μL recombinant human iduronidase (Aldurazyme, 0.58 mg/mL,Genzyme) were added to the reaction mixture and incubated overnight at37° C. The mixture was diluted in glycine buffer, pH 10.9, and released4-MU was quantified by fluorescence (excitation 365 nm, emission 450 nm)compared with standard dilutions of free 4-MU.

2. Serum Anti-IDS Antibodies

Blood for measurement of serum anti-hIDS antibodies was collected atterminal necropsy endpoints of study W2356 (Day 21) and study W2301(approximately 3 month) by cardiac puncture. Serum was separated andfrozen on dry ice and stored at −80° C. until analyzed. Polystyreneplates were coated overnight with recombinant human IDS (R&D Systems), 5μg/mL in PBS, titrated to pH 5.8. Plates were washed and blocked 1 hourin 2% bovine serum albumin (BSA) in neutral PBS. Plates were thenincubated with serum samples diluted 1:1000 in PBS. Bound antibody wasdetected with horseradish peroxidase (HRP)-conjugated goat anti-mouseantibody (Abcam) diluted 1:10,000 in PBS with 2% BSA. The assay wasdeveloped using tetramethylbenzidine substrate and stopped with 2Nsulfuric acid before measuring absorbance at 450 nm. Titers weredetermined from a standard curve generated by serial dilution of apositive serum sample arbitrarily assigned a titer of 1:10,000.

DNA was isolated from tissues of the high dose group using the QIAmp DNAMini Kit and vector genomes quantified by TaqMan PCR as previouslydescribed (Bell et al, 2006). Total cellular DNA was extracted fromtissues using a QIAamp DNA Mini Kit (Qiagen, Valencia, Calif., USA).Detection and quantification of vector genomes in extracted DNA wereperformed by real-time PCR (TaqMan Universal Master Mix, AppliedBiosystems, Foster City, Calif., USA) using primer and probe setstargeted to the rBG polyA sequence.

Forward primer: 5V-TTCCCTCTGCCAAAAATTATGG-3V,; SEQ ID NO: 16Reverse primer: 5V-CCTTTATTAGCCAGAAGTCAGATGCT-3V,; SEQ ID NO: 17 Probe:6FAM-ACATCATGAAGCCCC-MGBNFQ,. SEQ ID NO: 18

The PCR conditions were set at 100 ng total cellular DNA as template,300 nM primers, and 200 nM probes each. Cycles were for 10 min at 95 C,40 cycles of 15s at 95° C., and 1 min at 60° C. A value of 1×10⁴ genomecopies per 100 ng DNA was calculated to represent one genome copy percell.

H&E staining was performed on formalin-fixed paraffin-embedded rostralbrain sections from the 3 months pi necropsy, according to standardprotocols. H&E sections of the brain were evaluated for evidence oftoxicity by a board certified veterinary pathologist and the range offindings related to the MPS II phenotype were characterized.Subsequently, the pathologist re-examined the slides without knowledgeof treatment and scored the histologic manifestations of MPS IIphenotype including glial cytoplasmic vacuolation, neuronal cytoplasmicswelling and accumulation of amphiphilic material. The number of cellsstaining positive for LIMP2 and GM3 was quantified in 2-4 brain sectionsfrom each animal of W2356 study by trained GTP Morphology corepersonnel.

GM3 (Frozen Sections): GM3 immunostaining was performed on 30 um thickfloating cryosections as described using monoclonal antibody DH2(Glycotech, Gaithersburg, Md.) as primary antibody followed by abiotinylated secondary anti-mouse antibody (Jackson Immunoresearch, WestGrove, Pa.) and detection with a Vectastain Elite ABC kit (Vector Labs,Burlingame, Calif.). Stained sections were transferred onto glass slidesand mounted with Fluoromount G (Electron Microscopy Sciences, Hatfield,Pa.).

LIMP2 (Formalin-fixed Sections): LIMP2 immunostaining was performed on 6um sections from formalin-fixed paraffin-embedded brain tissue. Sectionswere deparaffinized through an ethanol and xylene series, boiled in amicrowave for 6 minutes in 10 mmol/L citrate buffer (pH 6.0) for antigenretrieval, and blocked with 1% donkey serum in PBS+0.2% Triton for 15minutes followed by sequential incubation with primary (1 hour) andlabeled secondary (45 minutes) antibodies diluted in blocking buffer.The primary antibody was rabbit anti-LIMP2 (Novus Biologicals,Littleton, Colo., 1:200) and the secondary antibody was FITC- orTRITC-labeled donkey anti-rabbit (Jackson Immunoresearch).

Tissue samples obtained as described above (section b) were homogenizedin lysis buffer (0.2% Triton-X100, 0.9% NaCl, pH 4.0) using aTissueLyser (Qiagen). Samples were freeze-thawed and clarified bycentrifugation. Protein was quantified by BCA assay. IDS activity wasmeasured using the fluorogenic substrate 4-methylumbelliferylα-L-idopyranosiduronic acid 2-sulfate (Santa Cruz Biotechnology).Hexosaminidase activity and GAG concentration were measured usingstandard procedures as described (Hinderer et al. 2015).

IV. Computerized Systems

For Open Field, data were entered into Excel and analyzed using GraphpadPrism. For Y-maze, data were entered into Excel and analyzed usingGraphpad Prism. For CFC, Freezescan, CleverSys Inc was used. For NOR,MATLAB Implementation and User Guide:www.seas.upenn.edu/˜molneuro/autotyping.html was utilized.

V. Statistical Analysis

Tissue GAG content, Hex activity and brain storage lesions in treatedand untreated mice were compared using a one-way ANOVA followed byDunnett's multiple comparisons test. Open field and Y-maze data wereanalyzed with Student's t-test. A two-way ANOVA and Dunnett's post-hocanalysis was applied to the fear conditioning data to assess trial andgenotype effects. For the novel object recognition test, time exploringthe novel object vs familiar object was compared using a t-test for eachgroup, followed by a Bonferroni correction for multiple comparisons.

VI. Results

No mortality was observed. There were no clinical observations that wereconsidered related to the treatment with the vector.

To evaluate general locomotor activity, open field test was performed.IDSγ/− mice showed normal exploratory activity in an open field arenacompared to wildtype littermates (FIGS. 9A-9C).

Comparison of wildtype and IDS γ/− (MPS II) mouse performance in OpenField Arena (Horizontal Movement) was performed (FIG. 9A). Spontaneousactivity during a 10 minutes trial was automatically recorded based onXY-axis beam breaks that capture horizontal movement. No difference wasseen between WT and MPS II mice.

Comparison of wildtype and IDS γ/− (MPS II) mouse performance in OpenField Arenas (Vertical Movement or Rearing) was performed (FIG. 9B).Spontaneous activity during a 10 minutes trial was automaticallyrecorded based on Z-axis beam breaks that capture rearing of the mice ontheir hind legs. No difference was seen between WT and MPS II mice.

Comparison of wildtype and IDS γ/− (MPS II) mouse performance in OpenField Arena (Center Activity) was performed (FIG. 9C). Spontaneousactivity during a 10 minutes trial was automatically recorded based oncenter beam breaks that capture time spent in open areas, as a marker ofanxiety. No significant difference was seen between WT and MPS II mice.

To evaluate short term memory, Y-Maze activity was analyzed. Comparisonof wildtype and IDS γ/− (MPS II) mouse during an 8-Minute Y-Maze TestingSession. No difference was seen in the total number of arm entriesbetween WT and MPS II mice. The IDSγ/− mice also had similar numbers ofarm entries and equivalent spontaneous alternations in the Y-mazecompared to wildtype littermates, indicating that the disease processdoes not affect short term memory (FIG. 9D).

To evaluate long term memory, contextual fear conditioning (FC) wasperformed. Evaluation of treatment effect on cognition using thecontextual fear conditioning was tested in wildtype, untreated, andtreated MPS II mice. Percent Freezing was plotted on y axis (FIG. 6B).Freezing behavior prior to learning (Pre) is compared to freezingbehavior after conditioning (Probe) for each group (FIG. 6B). In the FCassay, all mice (IDSγ/− and wildtype) showed an increase in the percenttime freezing during the recall phase of the test, demonstrating thatlearning had occurred, but IDSγ/− mice showed reduced freezing relativeto wild type littermates (data not shown). In mice treated withAAV9.CB7.CI.hIDS.rBG, there was no clear improvement in response tocontextual fear conditioning, although treatment-related effects weredifficult to evaluate due to the small difference between normal anduntreated IDSγ/− mice (FIG. 6B).

To further evaluate long term memory, Novel Object Recognition wasperformed. Evaluation of treatment effect on cognition using the NovelObject Recognition was tested in wildtype, untreated and treated MPS IIMice. Comparison of time spent exploring the novel object versus thefamiliar object for wildtype, untreated and treated IDS γ/− (MPS II)Mice was performed. Increased time spent exploring the novel object(showing memorization of the familiar object) was seen in all group oftreated mice but was statistically significant in the mid-dose grouponly (FIG. 6C). Wild type mice demonstrated a preference for a novelobject, as expected, but IDSγ/− did not show a preference showing a lackof memorization of the familiar object (long term memory impairment).Intrathecal AAV9 gene therapy produced improvements in the NOR deficitsobserved in the IDSγ/− (MPS II) mice but with no clear dose response.The preference for the novel object was statistically significant onlyin the mid-dose cohort, although the study was not sufficiently poweredto compare the relative degree of rescue of behavioral deficits amongdosing groups (FIG. 6C).

Dose-dependent increase in Brain IDS Activity (Day 21) in wildtype,untreated and treated MPS II mice was observed (FIG. 2B). Only the highdose group had levels similar to the wild-type at this early time point,which was expected as the expression had not reached its maximum yet.

Dose-dependent increase in IDS Activity (Day 90) in wildtype, untreatedand treated MPS II Mice was observed in CSF collected at necropsy 3months pi (FIG. 2A). Essentially no IDS activity was detected inuntreated IDS γ/− (MPS II) mice.

Furthermore, dose-dependent increase in serum IDS Activity (Day 90) inwildtype, untreated and treated MPS II Mice was detected at necropsy 3months pi (FIG. 2C). Essentially no activity was detected in serum ofuntreated IDS γ/− (MPS II) mice.

Three months pi, hexosaminidase activity (Day 90) was normalized in adose-dependent fashion in both liver (FIG. 4C) and heart (FIG. 4D) ofwildtype, untreated and treated MPS II Mice. This normalization of asecondary increased enzymatic activity showed restoration of lysosomalhomeostasis.

Hepatic and Cardiac GAG Storage in wildtype, untreated and treated MPSII mice was investigated. Three months pi, tissue content of GAG wasdose dependently reduced in both heart (FIG. 4B) and liver (FIG. 4A) andwas comparable to the wildtype tissue content at the mid and high doselevels. Liver was corrected at all doses and heart showed partialimprovement mostly at mid and high doses.

Furthermore, humoral immunogenicity represented by antibody responseagainst human IDS was evaluated in MPS II mice treated with ICVAAV9.CB7.CI.hIDS.rBG. Antibodies to human IDS were detected in serum ofseveral animals in the mid- and high-dose cohorts only at both Days 21and 90 (FIG. 8). At both timepoints, most animals had no detectablehumoral immune response (similar to control). About one third of theanimals in mid-dose and about 20% of the animals groups had antibodieslevels above the background. The response was less pronounced in thehigh dose group. FIG. 8 shows both necropsy endpoints data in aggregate.

There were no treatment-related histopathology findings in the brain.All microscopic findings present in this study were considered to beeither normal background for animals of this species, age, and sex,related to the disease model, or related to the vector administrationprocedure.

A scoring system based on cytoplasmic vacuolation was established andused for subsequent re-evaluation of all test animals without knowledgeof treatment or phenotype. Cytoplasmic vacuolation in glia,characterized by large clear vacuoles with eccentric or peripheraldisplacement of nuclei, was a distinctive feature of the IDSγ/−genotype. There was regional variability in the amount of vacuolation inthe brain regions that were evaluated. Neuronal accumulation ofamphiphilic material was less prominent and only observed in untreatedIDSγ/− mice. Treatment with AAV9.CB7.CI.hIDS.RBG decreased the amountand frequency of glial vacuolation and neuronal accumulation ofamphophilic material in all brain regions examined Cumulative pathologyscores demonstrate a treatment-related improvement in glial and neuronallesions (FIG. 27).

Immunohistochemical staining of GM3 and LIMP2 in wildtype, untreated andtreated MPS II Mice was performed. The brains of untreated IDS γ/− miceshowed clear histological evidence of lysosomal storage in neurons,including accumulation of the lysosomal membrane protein LIMP2, as wellas secondary storage of gangliosides including GM3 (FIGS. 5A to 5J).Images demonstrated that there was reduction of lysosomal storage at alldoses, with the high dose group animals being essentially similar to theWT controls (FIGS. 5A to 5J). Quantitation of dose-dependent reductionin GM3- and LIMP2-Positive Cells was performed in wildtype, untreatedand treated MPS II Mice. There was significant reduction of lysosomalstorage at all doses, with the high dose group animals being essentiallysimilar to the WT controls. Treated mice demonstrated dose-dependentdecreases in neuronal storage lesions, evidenced by reduced LIMP2 andGM3 staining (FIGS. 5K and 5L).

Biodistribution of AAV9.CB7.CI.hIDS.rBG in MPS II Mice from the highdose group (Day 21) was analyzed. Biodistribution data demonstrated thatthe brain tissue was transduced (1 to 10 GC per diploid genome), as wereall the peripheral tissues that were evaluated, especially the liver(FIG. 3). Concentrations in the liver and CNS were between 1 and 10GC/diploid genome; concentrations in lung between 0.1 and 1 GC/diploidgenome, and concentrations in heart and spleen less than 0.1 GC/diploidgenome.

VIII. Conclusion

ICV administration of AAV9.CB7.CI.hIDS.rBG to C57BL/6 IDS γ/− (MPS II)mice at up to 3×10¹⁰ GC (5.2×10¹⁰ GC ddPCR) was well tolerated, with noclinical signs or mortality, and resulted in distribution to the CNS aswell as to peripheral tissues, particularly liver, showing partialredistribution of injected viral particles from the CSF to theperipheral blood.

There was evidence of IDS gene expression in the brain as evidenced bydetection of the vector and dose dependent increases in IDS activity inthe brain at Day 21 and in the CSF 3 months pi, with enzymatic activityclose to wildtype level at the high dose (brain tissue) and comparableto higher than wildtype at the mid and high doses (CSF). There was dosedependent normalization of the lysosomal compartment, as shown byreductions in LIMP2 and GM3 staining in the CNS at all doses 3 monthspi. In H&E stained brain sections, dose dependent reductions in theamount and frequency of glial vacuolation and neuronal accumulation ofamphophilic material, indicators of MPS II CNS phenotype, were alsoobserved. Corresponding to the changes in CNS lysosomal content andimprovements in disease-related morphology in the H&E stained sections,there were improvements in one measure of long term memory (novel objectrecognition) but not in the other measure of long term memory,contextual fear conditioning. No clear dose response was apparent in theimprovement in NOR. Performance of MPS II mice in other tests of CNSfunction, the open field test and the Y-maze, was comparable to that ofwild type mice and therefore mice treated with vector were not evaluatedin these assays.

Dose dependent increases in serum IDS activity were also observed 3months pi with levels comparable to (low dose) or higher than the wildtype (mid/high doses). Reflecting the normalization of IDS activity inserum, the MPS II mice had dose dependent decreases in hexosaminidaseactivity and GAG content in the liver and heart; hepatic hexosaminidaseand GAG levels were normalized at all dose levels and cardiachexosaminidase and GAG activity was normalized at the mid and highdoses, respectively. The highly transduced liver (1 to 10 GC per diploidgenome) probably acted as a depot organ for the secretion of IDS in theserum and cross-correction of the heart at the higher doses.

There was no evidence of test-article related toxicity in the brain,although changes related to the ICV administration procedure itself wereobserved in some mice. Humoral immune response to the transgene wasminor, observed only in some mid- and high-dose animals without impacton the health or brain histopathology of those animals.

In conclusion, AAV9.CB7.CI.IDS.rBG was well tolerated in MPS II mice atall dose levels and resulted in dose-dependent increases in IDS levelsthat were associated with improvements in both CNS and peripheralparameters of MPS II.

The lowest dose administered, 3×10⁸ GC (5.2×10⁸ GC ddPCR), was theminimum effective dose in this study.

SEQUENCE LISTING FREE TEXT

The following information is provided for sequences containing free textunder numeric identifier <223>.

SEQ ID NO: (containing free text) Free text under <223> 1 <223> humanIDS enzyme 3 <223> CB7.CI.hIDS.RBG <220> <221> misc_feature <222> (2) .. . (131) <223> 5′ ITR <220> <221> promoter <222> (199) . . . (580)<223> CMV IE promoter <220> <221> promoter <222> (583) . . . (864) <223>CB promoter <220> <221> TATA_signal <222> (837) . . . (840) <220> <221>Intron <222> (959) . . . (1930) <223> chicken beta-actin intron <220><221> CDS <222> (1937) . . . (3589) <223> hIDS <220> <221> polyA_signal<222> (3623) . . . (3749) <223> rabbit globin polyA <220> <221>misc_feature <222> (3838) . . . (3967) <223> 3′ ITR 4 <223> SyntheticConstruct 5 <223> CB6.hIDS.IRES.SUMF1 <220> <221> misc_feature <222>(11) . . . (140) <223> 5′ITR <220> <221> enhancer <222> (208) . . .(589) <223> CMV IE enhancer <220> <221> promoter <222> (600) . . . (859)<223> CB promoter <220> <221> TATA_signal <222> (835) . . . (842) <220><221> Intron <222> (976) . . . (1108) <220> <221> CDS <222> (1177) . . .(2829) <223> hIDS <220> <221> misc_feature <222> (2830) . . . (3422)<223> IRES <220> <221> CDS <222> (3423) . . . (4547) <223> hSUMF <220><221> polyA_signal <222> (4620) . . . (4746) <223> rabbit globin polyA<220> <221> misc_feature <222> (4835) . . . (4964) <223> 3′ITR 6 <223>Synthetic Construct 7 <223> Synthetic Construct 8 <223>CB6.hIDSco.IRES.hSUMF1co <220> <221> misc_feature <222> (11) . . . (140)<223> 5′ITR <220> <221> enhancer <222> (208) . . . (589) <223> CMV IEenhancer <220> <221> promoter <222> (600) . . . (859) <223> CB promoter<220> <221> TATA_signal <222> (835) . . . (842) <220> <221> Intron <222>(976) . . . (1108) <220> <221> CDS <222> (1177) . . . (2829) <223>hIDSco <220> <221> misc_feature <222> (2830) . . . (3422) <223> IRES<220> <221> CDS <222> (3423) . . . (4553) <223> hSUMF1co <220> <221>polyA_signal <222> (4620) . . . (4746) <223> rabbit globin polyA <220><221> misc_feature <222> (4835) . . . (4964) <223> 3′ITR 9 <223>Synthetic Construct 10 <223> Synthetic Construct 11 <223> CB7.hIDSco.RBG<220> <221> misc_feature <222> (2) . . . (131) <223> 5′ITR <220> <221>promoter <222> (199) . . . (580) <223> CMV IE promoter <220> <221>promoter <222> (583) . . . (864) <223> CB promoter <220> <221>TATA_signal <222> (837) . . . (840) <220> <221> Intron <222> (959) . . .(1930) <223> chicken beta-actin intron <220> <221> CDS <222> (1937) . .. (3589) <223> hIDSco <220> <221> polyA_signal <222> (3623) . . . (3749)<223> rabbit globin polyA <220> <221> misc_feature <222> (3838) . . .(3967) <223> 3′ ITR 12 <223> Synthetic Construct 13 <223> AAV9 capsidamino acid sequence 14 <223> CB7.CI.hIDS.RBG <220> <221> misc_feature<222> (2) . . . (131) <223> 5′ITR <220> <221> promoter <222> (199) . . .(580) <223> CMV IE promoter <220> <221> promoter <222> (583) . . . (864)<223> CB promoter <220> <221> TATA_signal <222> (837) . . . (840) <220><221> Intron <222> (957) . . . (1928) <223> chicken beta-actin intron<220> <221> CDS <222> (1935) . . . (3587) <223> hIDS <220> <221>polyA_signal <222> (3621) . . . (3747) <223> rabbit globin polyA <220><221> misc_feature <222> (3836) . . . (3965) <223> 3′ITR 15 <223>Synthetic Construct 16 <223> synthesized sequence 17 <223> synthesizedsequence 18 <223> Synthesized sequence

All publications, patents, and patent applications cited in thisapplication are incorporated herein by reference in their entirety, asare U.S. Provisional Patent Application No. 62/452,494, filed Jan. 31,2017, U.S. Provisional Patent Application No. 62/367,780, filed Jul. 28,2016, U.S. Provisional Patent Application No. 62/337,163, filed May 16,2016, U.S. Provisional Application No. 62/330,938, filed May 3, 2016,U.S. Provisional Application No. 62/323,194, filed Apr. 15, 2016, andU.S. patent application Ser. No. 16/093,413, filed Oct. 12, 2018.Similarly, the SEQ ID NOs which are referenced herein and which appearin the appended Sequence Listing filed herewith under file number“UPN-16-7771PCT_ST25”, are hereby incorporated by reference. Althoughthe foregoing invention has been described in some detail by way ofillustration and example for purposes of clarity of understanding, itwill be readily apparent to those of ordinary skill in the art in lightof the teachings of this invention that certain changes andmodifications can be made thereto without departing from the spirit orscope of the appended claims.

1. A pharmaceutical composition suitable for intrathecal administrationin human subjects, comprising a suspension of replication deficientrecombinant adeno-associated virus (rAAV) in a formulation buffer,wherein: (a) the rAAV comprises a heterologous nucleic acid encodinghuman iduronate-2-sulfatase (hIDS) packaged in an AAV9 capsid; (b) theformulation buffer comprises a physiologically compatible aqueousbuffer, and optional surfactants and excipients; and (c) (i) the rAAVGenome Copy (GC) titer is at least 1.0×10¹³ GC/ml (+/−20%); and/or (ii)the rAAV Empty/Full particle ratio is at least about 80% free of emptycapsids; and/or (iii) a dose of at least about 2.5×10¹⁰ GC/g brain massto about 3.6×10¹¹ GC/g brain mass of the rAAV suspension has potency.